Arriving at a correlation between the flagellar arrangement and multicellularity

Cilia and flagella are organelles of motility that enable cells to swim or move liquid over its surface. An exhaustive literature survey for the presence of the organelle in organisms across phyla showed that most animal cells harbor cilia in contrast to very few fungal cells. While this was not unexpected, it was the position and arrangement of this organelle in each cell that intrigued our attention. Natural selection might have favored motility over chemotaxis;and it would have done so to evolve a stable structure that could have undergone an optimization process requiring a precise geometry in the shape of cells and the structure that would help cells to move. The positioning of such a structure would play a pre-dominant role in optimal motility. It is now known that the flagellar position of a cell is a genetically distinct trait, occasionally used in phylogeny of bacteria, distributed in distinguishing patterns over cellular surface, but basically are of two types, either polar (one flagellum arising from one pole per cell) or peritrichous (lateral flagella distributed over the entire cell surface). Irrespective of the cellular habitat, flagella origin, ultrastructure and proteome, the present investigation surveyed 26 sub-types of flagellar arrangements from as many species as possible. A peculiar pattern ensued-Prokaryotes harbored predominantly polar and peritrichous types;eukaryotes showed a mere change of the peritrichous one. These numbers when used to create a Similarity tree depicted a similarity distance of 14 between the Eubacteria and Archaebacteria forming the first neighborhood;Protozoans, Algae, Fungi, Plantae and Animalia formed a second neighborhood. We offer a working hypothesis for this pattern and the gradual shift in the flagellar arrangement from polar, peritrichous, sub-apical, and apical to lateral throughout evolution.

Macroalgal deep genomics illuminate multiple paths to aquatic,photosynthetic multicellularity

Macroalgae are multicellular,aquatic autotrophs that play vital roles in global climate maintenance and have diverse applications in biotechnology and eco-engineering,which are directly linked to their multicellularity phenotypes.However,their genomic diversity and the evolutionary mechanisms underlying multicellularity in these organisms remain uncharacterized.In this study,we sequenced 110 macroalgal genomes from diverse climates and phyla,and identified key genomic features that distinguish them from their microalgal relatives.Genes for cell adhesion,extracellular matrix formation,cell polarity,transport,and cell differentiation distinguish macroalgae from microalgae across all three major phyla,constituting conserved and unique gene sets supporting multicellular processes.Adhesome genes show phylum-and climate-specific expansions that may facilitate niche adaptation.Collectively,our study reveals genetic determinants of convergent and divergent evolutionary trajectories that have shaped morphological diversity in macroalgae and provides genome-wide frameworks to understand photosynthetic multicellular evolution in aquatic environments.

Simulating traumatic brain injury in vitro:developing high throughput models to test biomaterial based therapies

Traumatic brain injuries are serious clinical incidents associated with some of the poorest outcomes in neurological practice.Coupled with the limited regenerative capacity of the brain,this has significant implications for patients,carers,and healthcare systems,and the requirement for life-long care in some cases.Clinical treatment currently focuses on limiting the initial neural damage with longterm care/support from multidisciplinary teams.Therapies targeting neuroprotection and neural regeneration are not currently available but are the focus of intensive research.Biomaterial-based interventions are gaining popularity for a range of applications including biomolecule and drug delive ry,and to function as cellular scaffolds.Experimental investigations into the development of such novel therapeutics for traumatic brain injury will be critically underpinned by the availability of appropriate high thro ughput,facile,ethically viable,and pathomimetic biological model systems.This represents a significant challenge for researchers given the pathological complexity of traumatic brain injury.Specifically,there is a concerted post-injury response mounted by multiple neural cell types which includes microglial activation and astroglial scarring with the expression of a range of growth inhibito ry molecules and cytokines in the lesion environment.Here,we review common models used for the study of traumatic brain injury(ranging from live animal models to in vitro systems),focusing on penetrating traumatic brain injury models.We discuss their relative advantages and drawbacks for the developmental testing of biomaterial-based therapies.

Ultrastructure of the Multicellular Nodules in Hypericum perforatum Leaves

With the development of the multicellular nodules in the leaves of Hypericum per plastids increased both in number and volume, and some plastids degenerated and were surrounded by dark tubular elements and vesicles. Some vesicles fused with vacuoles and secreted substances into vacuoles. There are many multivesicles, multimembrane structures and osmiophilic droplets in vacuoles of nodule cells. Meanwhile, dictyosome secreted vesicles into the vacuoles. However, there is a large central vacuole completely filled with secretory materials (hypericin) in matured multicellular nodules. This suggests that the osmiophilic droplets may be a precursor of hypericin originated from the degenerated plastids. There were abundant endoplasmic reticulums and dictyosomes between the plastids and osmiophilic droplets, suggesting that they may be involved in the synthesis and/or transport of the precursor of hypericin.

Into the life and death: RecA a WISE factor working to integrate survival and evolution in <i>Escherichia coli</i>

Escherichia coli RecA has been considered traditionally a cellular protein with multiple vital functions working to ensure the maintenance of integrity of genome in each individual bacterial cell as well as promoting swarming migration in collectivity. On the contrary, recently it has been described that RecA promotes cellular apoptotic-like death (ALD), a pathway of programmed cellular death (PCD). In fact, RecA has been dubbed as the major apoptotic executor in E. coli. From these studies, RecA emerges as a prototypical Gin/Gan protein that despite of their intrinsic vital and lethal anfi-funcionality becomes in a WISE factor: a Worker to Integrate Survival and Evolution in E. coli evolving populations living in community. Here, I provide a review of recent experimental and conceptual advances trying to understand these RecA’s antagonistic roles in appearance contradictory under a unified biological vision.

occurrence and microscopic analyses of multicellular magnetotactic prokaryotes from coastal sediments in the Yellow Sea

Multicellular magnetotactic prokaryotes （MMPs） are a group of aggregates composed of 7-45 gram-negative cells synthesizing intracellular magnetic crystals. Although they are thought to be globally distributed, MMPs have been observed only in marine environments in America and Europe. Most MMPs share a rosette-like morphology and biomineralize iron sulfide crystals. In the present study, abundant MMPs were observed, with a density of 26 ind./cm^3, in the sediments of a coastal lagoon, Lake Yuehu, in the Yellow Sea. Optical microscopy showed that all of them were rosette shaped with a diameter of 5.5±0.8 μm. Transmission electron microscopy revealed that these MMPs were composed of 10- 16 ovoid cells and flagellated peritrichously. High-resolution transmission electron microscopy and energy dispersive X-ray analysis indicated that they biomineralized bullet-shaped magnetite crystals in highly organized parallel chains within which the magnetosomes were oriented in the same direction. This is the first report of MMPs from Asia and demonstrates the ubiquitous distribution of MMPs.

Chinese medicinal compound delisheng has satisfactory anti-tumor activity,and is associated with up-regulation of endostatin in human hepatocellular carcinoma cell line HepG2 in three-dimensional culture

AIM： TO investigate the multicellular resistance of human hepatocellular carcinoma HepG2 cells in three-dimensional culture to delisheng, 5-fluorouracil and adriamycin, and the possible molecular mechanisms of delisheng. METHODS： Human hepatocellular carcinoma HepG2 cells were cultured with a liquid overlay technique. After the formation of multicellular spheroids, morphology was analyzed by phase contrast microscopy, scanning electron microscopy and transmission electron microscopy. Sensitivity of HepG2 cells to delisheng, 5-fluorouracil and adriamycin was investigated by Ml-I- assay in multicelluar spheroids and monolayers. Vascular endothelial growth factor （VEGF） and endostatin expression were analyzed in multicellular spheroids treated with delisheng, 5-fluorouracil, adriamycin and negative control PBS, with immunohistochemical staining. RESULTS： Multicellular spheroids exhibited structural characteristics somewhat different to those in monolayers. The cells in three-dimensional cell culture turned out to be less sensitive to delisheng, 5-fluorouracil and adriamycin than the cells cultured in monolayer. This showed that delisheng had a satisfactory cells inhibition ratio compared to 5-fluorouracil and adriamycin. Immunohistochemical staining showed that VEGF and endostatin expression was positive during growth as multicellular spheroids, and endostatin expression in spheroids with treatment of delisheng was higher than that with 5-fluorouracil, adriamycin and PBS （139.35 ± 7.83, 159.23 ± 10.34, 162.83 ± 3.47 and 148.48 ± 11.06, P 〈 0.05）.CONCLUSION： Chinese medicine compound delisheng has satisfactory anti-tumor activity in HepG2 cells in three-dimensional culture, and the effects are associated with up-regulation of endostatin.

Malignant peritoneal effusion acting as a tumor environment in ovarian cancer progression: Impact and significance

Until recently, ovarian cancer research has mainly focused on the tumor cells themselves ignoring for the most part the surrounding tumor environment which includes malignant peritoneal effusions. However, one of the major conceptual advances in oncology over the last few years has been the appreciation that cancer progression cannot be explained by aberrations in cancer cells themselves and is strongly influenced by the surrounding tumor environment. The mechanisms of ovarian cancer progression differ from that of other solid tumors because ovarian cancer cells primarily disseminate within the peritoneal cavity.Malignant peritoneal effusion accumulates in the peritoneal cavity during ovarian cancer progression. These exudative fluids act as a unique tumor environment providing a framework that orchestrates cellular and molecular changes contributing to aggressiveness and disease progression. The composition of ascites, which includes cellular and acellular components, constantly adapts during the course of the disease in response to various cellular cues originating from both tumor and stromal cells. The tumor environment that represents peritoneal effusions closely constitute an ecosystem, with specific cell types and signaling molecules increasing and decreasing during the course of the disease progression creating a single complex network. Although recent advances aiming to understand the ovarian tumor environment have focused one at a time on components, the net impact of the whole environment cannot be understood simply from its parts or outside is environmental context.

Isolated Cells of Porphyra yezoensis Cultured on Solid Medium

Vegetative cells of Porphyra yezoensis are isolated with sea snail enzyme and cultured on the solidified agar medium. The results of experiments show that the isolated cells can survive,divide and regenerate well on the medium solidified with agar. The first division on the solid medium starts after 7 days' culture, 4 days later than the liquid culture. The survival rate of isolated cells is 71.3% on the solid medium, lower than the 86.2% of that in seawater.Thalli, thalloids,conchocelis, spermatangia and multicellular masses are developed on the solid/medium in the first month, slowly but normally. Spermatangia sacs disappear within 4 weeks. Without adding nutrient liquid onto the surface of solid medium or injecting seawater under the agar layer in order to keep moisture, the thalli and cell groups release monospores to form new thalli instead of enlarging their areas after 5 weeks' culturing. Some monospores regenerate new thalli. Other monospores lose their pigments and minimize their volume and divide quickly to form light pink calli. After 16 weeks, numerous calli can be seen on the solid medium and after 24 weeks' culturing, almost only calli and conchocelis can be seen. If the calli are immersed in seawater, the monospores are released and may develop into young thallus.

Cytoskeleton reorganization and ultrastructural damage induced by gliadin in a three-dimensional in vitro model

AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure （by means of light microscopy and scanning electron microscopy）, and the effect of gliadin on actin （phalloidin fluorescence） and the tight-junction protein occludin and zonula occluden-1.RESULTS： The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gUadin decreased the number of intracellular actin filaments, impaired and disassembled the integrity of the tight-junction system.CONCLUSION： Our data obtained from an ＂in vivolike＂ polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions.

Role of soluble factors and three-dimensional culture in in vitro differentiation of intestinal macrophages

AIM: To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. METHODS: To test whether soluble or membrane bound factors induce IMAC-differentiation, freshly elutriated monocytes (MO) were incubated with conditioned media or cell membranes of intestinal epithelial cells (IEC) or cultured with IEC in transwell systems. To determine the importance of an active migration of MO, three- dimensional aggregates from a 1:1-mixture of MO and IEC were examined by immunohistochemistry and flow cytometry. Apoptosis was examined by caspase-3 Western blots. Extracellular matrix production in differentiation models was compared by immunohistochemistry. RESULTS: IMAC differentiation was observed in a complex three-dimensional co-culture model (multicellular spheroid, MCS) with IEC after migration of MO into the spheroids. By co-culture of MO with conditioned media or membrane preparations of IEC no IMAC differentiation was induced. Co-culture of MO with IEC in transwell- cultures, with the two cell populations separated by a membrane also did not result in intestinal-like differentiation of MO. In contrast to IEC-spheroids with immigrating MO in mixed MCS of IEC and MO only a small subpopulation of MO was able to survive the seven day culture period. CONCLUSION: Intestinal-like differentiation of MO in vitro is only induced in the complex three-dimensional MCS model after immigration of MO indicating a roleof cell-matrix and/or cell-cell interactions during the differentiation of IMACs.

Elucidating the Uptake and Distribution of Nanoparticles in Solid Tumors via a Multilayered Cell Culture Model

Multicellular layers(MCLs) have previously been used to determine the pharmacokinetics of a variety of different cancer drugs including paclitaxel, doxorubicin, methotrexate, and 5-fluorouracil across a number of cell lines. It is not known how nanoparticles(NPs) navigate through the tumor microenvironment once they leave the tumor blood vessel.In this study, we used the MCL model to study the uptake and penetration dynamics of NPs. Gold nanoparticles(GNPs)were used as a model system to map the NP distribution within tissue-like structures. Our results show that NP uptake and transport are dependent on the tumor cell type. MDA-MB-231 tissue showed deeper penetration of GNPs as compared to MCF-7 one. Intracellular and extracellular distributions of NPs were mapped using Cyto Viva imaging. The ability of MCLs to mimic tumor tissue characteristics makes them a useful tool in assessing the efficacy of particle distribution in solid tumors.

Hepatitis B virus infection modeling using multi-cellular organoids derived from human induced pluripotent stem cells

Chronic infection with hepatitis B virus(HBV)remains a global health concern despite the availability of vaccines.To date,the development of effective treatments has been severely hampered by the lack of reliable,reproducible,and scalable in vitro modeling systems that precisely recapitulate the virus life cycle and represent virus-host interactions.With the progressive understanding of liver organogenesis mechanisms,the development of human induced pluripotent stem cell(iPSC)-derived hepatic sources and stromal cellular compositions provides novel strategies for personalized modeling and treatment of liver disease.Further,advancements in three-dimensional culture of self-organized liver-like organoids considerably promote in vitro modeling of intact human liver tissue,in terms of both hepatic function and other physiological characteristics.Combined with our experiences in the investigation of HBV infections using liver organoids,we have summarized the advances in modeling reported thus far and discussed the limitations and ongoing challenges in the application of liver organoids,particularly those with multi-cellular components derived from human iPSCs.This review provides general guidelines for establishing clinical-grade iPSC-derived multi-cellular organoids in modeling personalized hepatitis virus infection and other liver diseases,as well as drug testing and transplantation therapy.

Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike induced pluripotent stem cells,they are cost-effective and non-tumorigenic.The utilization of AECs in regenerative medicine,however,is in its infancy.A general profile for AECs has not been comprehensively analyzed.Moreover,no hepatic differentiation protocol for AECs has yet been established.To this end,we independently compiled human AEC libraries,purified amniotic stem cells(ASCs),and co-cultured them with mesenchymal stem cells(MSCs)and human umbilical vein endothelial cell(HUVECs)in a 3D system which induces functional hepatic organoids.AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs,MSCs,and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients.Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry,Alkaline phosphatase(AP)staining,and flow cytometry.An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay.AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics.The 2D and 3D culture were compared by relative gene expression using several differentiation protocols.ASCs,MSCs,and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging,periodic acid Schiff,and an indocyanine green(ICG)test.RESULTS AECs have certain stemness markers such as EPCAM,SSEA4,and E-cadherin.One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers.Moreover,it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage.Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells.TJP1,associated with epidermal growth factor receptor,and MET,associated with hepatocyte growth factor receptor,were upregulated and may be important for hepatic differentiation.In conventional flat culture,the cells turned unviable and did not readily differentiate into hepatocytes.In 3D culture,however,hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol.Furthermore,the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity,hepatic-like glycogen storage,and ICG absorption/elimination.CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness.Under a 3D co-culture system,functional hepatic organoids were generated in a multicellular microenvironment.

Construction of Three-dimensional In Vitro Culture Model of Ovarian Carcinoma and the Study of Its Multicellular Drug Resistance

To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids （MCS） were obtained from three-dimensional culture. Drug sensitivity of monolayer cells （MC） and MCS were respectively tested by MTT staining and cytometry. The apoptosis of MC and MCS were determined by the flow cytometry （FCM）. The expression of bcl-2 and caspase-3 in A2780/MC and A2780/MCS were detected by using Western blot and caspase-3 assay kit, A2780/MC was compacted into mass after 2 days in three-dimensional cell culture model, and MCS had more than two layers of cells growing within 5 days. Compared with A2780/MC, A2780/MCS were more resistant to the anticancer drug, and the apoptosis rate was significantly lower than those of A2780/MC, The activity of caspase-3 in A2780/MCS was significantly lower than the A2780/MC. But the expression of bcl-2 in A2780/MCS was significantly higher than that in A2780/MC. It was suggested that the drug resistance of MCS might be associated with the overexpression of anti-apoptosis protein bcl-2 and the down-regulation of caspase-3 activity.

The vertical structure of the vegetative canopy of the brown algae Cystoseira(Black Sea)

In the coastal ecosystems of the Black Sea,macrophytobenthos and,in particular,the association of Cystoseira crinite,C.barbara,Cladostephus verticillatus,and Corallina mediterranea,with its thick vegetative canopy(VC),is the key contributor to primary production(PP).Though the vertical structure of the canopy,formed by the algal association,is of principal importance to the PP level,this subject has been long-neglected by researchers.The goal of our work was to compare vertical structures of the vegetative canopy of Cystoseira brown algae under diverse hydrodynamical conditions of the Crimean Peninsula coast.Samples were collected using the 50 cm×50 cm counting frame at eight stations positioned in shallow(55-60 cm deep)sites of Sevastopol Bay(Crimean Peninsula).Dry weight biomass of the VC was determined for all algae assemblage and for each algal species individually,per horizontal surface unit,at each height(Z).The study shows that:1)the VC is characterized by unimodal vertical distribution of biomass,with maximum estimate in the lower part,where the biomass increases to 85%of the total biomass;2)a series of single-peaked curves reliably describes the unimodal distribution of the biomass;thalli of different age groups are found along the canopy profile;and 3)algae found in epiphytic synusia prefer inhabiting the upper part of the VC.The role of environmental factors(seawater turbulence and solar radiation)is discussed in reference to the formation of the vertical structure,made up of the associations of the brown algae Cystoseira.

Damaging effects of gliadin on three-dimensional cell culture model

AIM： To evaluate the effects of gliadin on the oxidative environment in the “in vivo-like”model of a three-dimensional cell culture system. METHODS： LoVo cell line （intestinal adenocarcinoma） multicellular spheroids were treated with digested gliadin （with albumin used as a control）. Spheroid volumes, cell viability and morphology, lactate dehydrogenase （LDH） release, content of reduced glutathione （GSH） and activity of GSH-related enzymes were examined. The data were statistically analyzed using the Student＇s t-test （P〈0.05）. was considered statistically significant. RESULTS： Gliadin reduced cell viability （from 20% to 60%） and led to morphological alterations characterized by apoptotic findings and cytoskeletal injuries. LDH activity increased. The content of GSH reduced （-20% vscontrols）, and activity of GSH-related enzymes was significantly inhibited. CONCLUSION： Gliadin treatment induces an imbalance in the antioxidative mechanism of cells cultured by the three-dimensional technique. This alteration may explain cell damage directly caused by gliadin and the subsequentmorphological abnormalities.

DIFFERENTIAL EXPRESSION OF ADHESION MOLECULES (CD44,CD29,ICAM-1 AND E-CADHERIN) IN OVARIAN CANCER SK-OV-3ip1 CELLS GROWN AS MONOLAYER AND MULTICELLULAR AGGREGATES

Objective: To detect mRNA levels and expression ofCD44, CD54, CD29 and E-cadherin (E-cad) and to discuss their relationship with formation and drug resistance ofovarian cancer SKOV3ip1 multicellular aggregates.Methods: Liquid overlay system was employed to obtainmulticellular aggregates. mRNA levels and expression ofCD44, CD54, CD29 and E-cad were investigated with RTPCR and flow cytometry (FCM) respectively. Results:Compared with monolayer cells, RT-PCR results showed a decrease in CD44 mRNA level by 0.626-fold and a decrease in CD29 mRNA level by 0.792-fold in multicellularaggregates. However, an increase in CD54 mRNA level by 1.815-fold and an increase in E-cadherin mRNA level by1.344-fold were found in multicellular aggregates. Theresults revealed the downregulation of CD44 and CD29 and the upregulation of CD54 and E-cad genes activity. CD44 expression in monolayer cells and multicellular aggregates were 75.995?.046 and 50.700?.351 (%) respectively andthere was a significant decrease in multicellular aggregates (P=0.001). Compared with control cells, no expression of CD54 was detected in monolayer cells (P=0.563) but markedly elevated CD54 expression was detected in multicellular aggregates (15.780?.217) (%) (P<0.01). High expression of CD29 was seen in monolayer cells and also in multicellular aggregates with positive rates of 96.290+1.201 (%) and 92.494?.055 (%). However, the expression of CD29 in multicellular aggregates was significantly reduced (P=0.014). Also no expression of E-cadherin was found in monolayer cells compared with control cells (4.490?.283) (%) (P=0.65) while significantly increased expression in aggregates cells (17.258?5.572) (%) (P=0.003) was observed. Conclusion: Significant differences in mRNA levels and expression of CD44, CD54, CD29 and E-cadherin clearly exist between monolayer cells and multicellular aggregates, which may be associated with the formation of multicellular aggregates and its drug resistance.

3D Printing of Cell-Container-Like Scaffolds for Multicell Tissue Engineering

The development of an engineered non-contact multicellular coculture model that can mimic the in v iv o cell microenvironment of human tissues remains challenging.In this study,we successfully fabricated a cell-container-like scaffold composed of p-tricalcium phosphate/hydroxyapatite(p-TCP/HA)bioceramic that contains four different pore structures,including triangles,squares,parallelograms,and rectangles,by means of three-dimensional(3D)printing technology.These scaffolds can be used to simultaneously culture four types of cells in a non-contact way.An engineered 3D coculture model composed of human bone-marrow-derived mesenchymal stem cells(HBMSCs),human umbilical vein endothelial cells(HUVECs),human umbilical vein smooth muscle cells(HUVSMCs),and human dermal fibroblasts(HDFs)with a spatially controlled distribution was constructed to investigate the individual or synergistic effects of these cells in osteogenesis and angiogenesis.The results showed that three or four kinds of cells cocultured in 3D cell containers exhibited a higher cell proliferation rate in comparison with that of a single cell type.Detailed studies into the cell-cell interactions between HBMSCs and HUVECs revealed that the 3D cell containers with four separate spatial structures enhanced the angiogenesis and osteogenesis of cells by amplifying the paracrine effect of the cocultured cells.Furthermore,the establishment of multicellular non-contact systems including three types of cells and four types of cells,respectively,cocultured in 3D cell containers demonstrated obvious advantages in enhancing osteogenic and angiogenic differentiation in comparison with monoculture modes and two-cell coculture modes.This study offers a new direction for developing a scaffold-based multicellular non-contact coculture system for tissue regeneration.

MULTICELLULAR-MEDIATED RESISTANCE TO CISPLATIN AND TAXOL IN HUMAN OVARIAN CANCER SK-OV-3IP1 MULTICELLULAR AGGREGATES

Objective: To investigate the chemosensitivity of ovarian cancer SK-OV-3ip1 multicellular aggregates (MCA) to cisplatin and taxol and to explore the possible mechanisms. Methods: Liquid overlay system was employed to obtain MCA. We detected the resistance using trypan blue exclusion testing, clonogenic assay, cell cycle profiles and apoptosis with flow cytometry (FCM). Results: After cisplatin exposure, MCA cells showed nearly equal cell viability with monolayer cells (P=0.05). After 40μM cisplatin exposure for 12 h, no clone (≥50 cells) was formed, but more viable cells attached to the bottom of 24-well plate in MCA group than monolayer. Furthermore, apoptosis rate and cell cycle profiles with FCM had no significant change between MCA and monolayer cells. After taxol exposure, however, trypan blue exclusion testing demonstrated higher cell viability in MCA cells (P=0.003) and higher clone formation rate in 100-cell group than monolayer cells (0.01<P<0.025). No significant difference was found in 50-cell or 200-cell group but more viable cells in MCA group were observed. Taxol exposure caused significantly decreased apoptosis rate in MCA cells than monolayer cells (P=0.012). Taxol induced significant cell arrest at G2-M phase in monolayer cells (P=0.001), but abrogation of G2-M arrest was observed in MCA cells (P=0.002). Conclusion: Compared with monolayer cells, MCA cells from the same SK-OV-3ip1 cell line appear to be more resistant to taxol but not to cisplatin. Cell cycle redistribution and multicellular-mediated inhibition of apoptosis can partially account for the resistance.