Xerostomia (dry mouth) is frequently experienced by patients treated with radiotherapy for head and neck cancers or with Sjögren's syndrome, with no permanent cure existing for this debilitating condition. To this end, in vitro platforms are needed to test therapies directed at salivary (fluid-secreting) cells. However, since these are highly differentiated secretory cells, the maintenance of their differentiated state while expanding in numbers is challenging. In this study, the efficiency of three reversible thermo-ionically crosslinked gels: (1) alginate-gelatin (AG), (2) collagen-containing AG (AGC), and (3) hyaluronic acid-containing AG (AGHA), to recapitulate a native-like environment for human salivary gland (SG) cell expansion and 3D spheroid formation was compared. Although all gels were of mechanical properties comparable to human SG tissue (~11 kPa) and promoted the formation of 3D spheroids, AGHA gels produced larger (>100 cells/spheroid), viable (>93%), proliferative, and well-organized 3D SG spheroids while spatially and temporally maintaining the high expression of key SG proteins (aquaporin-5, NKCC1, ZO-1, α-amylase) for 14 days in culture. Moreover, the spheroids responded to agonist-induced stimulation by increasing α-amylase secretory granules. Here, we propose alternative low-cost, reproducible, and reversible AG-based 3D hydrogels that allow the facile and rapid retrieval of intact, highly viable 3D-SG spheroids.
Humans ; Hydrogels/chemistry* ; Acinar Cells/cytology* ; Spheroids, Cellular/cytology* ; Salivary Glands/cytology* ; Gelatin/chemistry* ; Collagen/chemistry* ; Alginates/chemistry* ; Cell Culture Techniques/methods* ; Hyaluronic Acid/chemistry* ; Cell Proliferation ; Cell Survival ; Cells, Cultured

OBJECTIVETo imitate tumor microenvironment in vivo through construction of two-layer cell spheroid as a three-dimensional tumor model, and to validate its application in the study of Cx43 expression in colorectal cancer cell-fibroblast interactions and colorectal cancer progression.

METHODSThe two-layer cell spheroid was constructed from SW620 colorectal cancer cells and HELF fibroblasts. The expression of Cx43 in the spheroid was detected by immunocytochemistry. The expression of Cx43 in cultured SW620 cells with or without co-cultured fibroblasts was detected by immunocytochemistry and immunofluorescence. The expression of Cx43 in colorectal cancer tissue was detected by immunohistochemistry.

RESULTSThe spheroid showed well-defined cellular morphology and clear boundary between two cell lines.Significant expression of Cx43 was found along the boundary.SW620 cells had no expression of Cx43 when cultured alone, while the expression of Cx43 was induced upon co-culturing with fibroblasts.In the colorectal cancer tissue, expression of Cx43 was minimal in the centre of tumor in contrast to an upregulated expression at invasive front.

CONCLUSIONSThe two-layer cell spheroid is an observable and sensitive model for cell-cell interaction for studies of tumor microenvironment.It can simulate colorectal cancer cell-fibroblast interactions through up-regulation of Cx43 expression.

Cell Communication ; Cell Line, Tumor ; Coculture Techniques ; Colorectal Neoplasms ; metabolism ; pathology ; Connexin 43 ; metabolism ; Fibroblasts ; cytology ; Humans ; Spheroids, Cellular ; cytology ; metabolism ; Tumor Microenvironment ; Up-Regulation

Hereditary diffuse leukoencephalopathy with neuroaxonal spheroids (HDLS) is a rare autosomal dominant leukoencephalopathy disease, and colony stimulating factor 1 receptor (CSF1R) is the only gene in which mutations are known to cause HDLS. HDLS should be suspected in individuals with progressive neurological decline, characteristic MR imaging findings, and positive family history. This article reviews recent advance in imaging findings, clinical manifestations, genetic counseling and management in HDLS.
Brain ; diagnostic imaging ; Humans ; Leukoencephalopathies ; diagnosis ; diagnostic imaging ; genetics ; physiopathology ; Radiography ; Spheroids, Cellular ; cytology

OBJECTIVE: To investigate the relationship between matrix matrix metalloproteinases (MMP)-9 expression in HCE1/multicellular spheroids (MCS) and invasion of cervical carcinoma, and to explore the effect of MMPs inhibitor GM6001 on the model that HCE1/MCS invade live human umbilical vein endothelium cell (HUVEC). METHODS: We established the invasion model by coculturing HCE1/MCS and HUVEC, and detected the MMP-9 expression in HCE1 monolayer cells (HCE1/MC), HCE1/MCS and HUVEC using immunohistochemistry and treated the HCE1/MCS invasion model with GM6001. RESULTS: A cervical squamous carcinoma cell HCE1 infiltrating model was established. Compared with that in HCE1/MC, MMP-9 expression in the HCE1/MCS was significantly higher (P<0.05). Compared with the control group, the invasion of HCE1/MCS was inhibited by 26.09% and 92.95% (P<0.05) in the GM6001 2.5 μmol/L and 12.5 μmol/L group. MMP-9 expression in HCE1/MCS in the GM6001 12.5 μmol/L group was obviously lower than that in the control group (P<0.05). With the increase of the concentration of GM6001, the inhibition increased. CONCLUSION The enhancement of HCE1/MCS invasion may be related to the up-regulation of MMP-9 expression in HCE1/MCS. GM6001 can down regulate the MMP-9 expression in HCE1/MCS and partially block the HCE1/MCS invasion to HUVEC.
Carcinoma, Squamous Cell ; pathology ; Cell Line ; Cell Line, Tumor ; Dipeptides ; pharmacology ; Endothelial Cells ; pathology ; Female ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Spheroids, Cellular ; metabolism ; pathology ; Umbilical Veins ; cytology ; Uterine Cervical Neoplasms ; pathology

OBJECTIVEIsolation and expansion tumor spheres from colorectal cancer cell line Colo205 cultured in serum-free medium(SFM) supplemented with human recombinant EGF and bFGF.

METHODSColo205 cells were cultivated in SFM,while cells cultivated in serum-supplemented medium(SSM) served as the control. Cells morphology were observed by optical microscope, and expression of intestinal stem cells marker Musashi-1 was detected by immunocytochemical. To induce cell differentiation, tumour spheres were cultivated without EGF and bFGF in the presence of 10% serum. Then we analysed expressions of stem cell surface markers CD133 and CD44 among undifferentiated cell, post-differentiated cells and routine Colo205 cells under serum-supplemented culture condition by flow cytometry. At last we compared cell cycle and spectral karyotype between two groups.

RESULTSIn SFM consisting of EGF and bFGF, a minority of Colo205 cells could survive, proliferate and form the suspended tumor spheres. We detected high Musashi-1 expression in these cells. Compared with the SSM group and the post-differentiation SFM group, the expressions of CD133 and CD44 were significantly increased in the undifferentiated SFM group (P<0.05). There was no statistical difference in the expression of CD133 and CD44 between the post-differentiation SFM group and the SSM group (P>0.05). Cell cycle analysis indicated that tumor spheres were of a high proliferation state.We could not find any noticeable difference in the number of chromatosomes between the SFM group and the SSM group.

CONCLUSIONTumor spheres in which enriched cancer stem cells can be generated under serum-free culture condition with EGF and bFGF.

AC133 Antigen ; Antigens, CD ; metabolism ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; Cell Proliferation ; Culture Media, Serum-Free ; Glycoproteins ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Neoplastic Stem Cells ; cytology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Peptides ; metabolism ; RNA-Binding Proteins ; metabolism ; Spheroids, Cellular ; cytology

When the size of a neurosphere cultured in vitro reaches a certain critical value, a necrotic core will appear inside the neurosphere because of the limitation of oxygen or other nutrients transport from medium to the cells in the neurasphere. Large necrotic core will greatly reduce the expansion of NSCs. The cellular automaton (CA) model is applied in this article to model the growth of NSCs in sphere state. The appearance and enlargement of the necrotic core in a neurosphere is calculated by coupling the CA model with the nutrient diffusion analysis in bioreactors. The calculation results indicate that the culture conditions, such as seeding density, the concentration of nutrients in medium and the mass transfer coefficient between a neurosphere and medium, have some effects on the appearance of the necrotic core. However, the necrotic core mainly depends on the inner diffusion. It will certainly appear if the size of the neurosphere is large enough even the outside mass transfer is in a good condition in bioreactors. Additionally, the appearance of the necrotic core resulting from the shortage of oxygen is earlier than that caused by the limitation of glucose. And the growth of the necrotic core is very fast after its appearance, and the whole neurosphere may become necrotic. The model developed with cellular automaton and mass transfer is a good qualitative representation of NSCs growth in bioreactors.
Bioreactors ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Computer Simulation ; Models, Biological ; Neurons ; cytology ; Spheroids, Cellular ; Stem Cells ; cytology ; Tissue Engineering ; methods