Engineering a vascularised 3D in vitro model of cancer progression.

The hallmark of tumours is the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues, which include hypoxia, also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. We engineered compartmentalized biomimetic colorectal tumouroids with stromal surrounds that comprised a range of ECM densities, composition and stromal cell populations. Recapitulating tissue ECM composition and stromal cell composition enhanced cancer cell invasion. Manipulation of ECM density was associated with an altered migration pattern from glandular buds (cellular aggregates) to epithelial cell sheets. Laminin appeared to be a critical component in regulating endothelial cell morphology and vascular network formation. Interestingly, the disruption of vascular networks by cancer cells was driven by changes in expression of several anti-angiogenic genes. Cancer cells cultured in our biomimetic tumouroids exhibited intratumoural heterogeneity that was associated with increased tumour invasion into the stroma. These findings demonstrate that our 3D in vitro tumour model exhibits biomimetic attributes that may permit their use in studying microenvironment clues of tumour progression and angiogenesis.

Optimising contraction and alignment of cellular collagen hydrogels to achieve reliable and consistent engineered anisotropic tissue.

Engineered anisotropic tissue constructs containing aligned cell and extracellular matrix structures are useful as in vitro models and for regenerative medicine. They are of particular interest for nervous system modelling and regeneration, where tracts of aligned neurons and glia are required. The self-alignment of cells and matrix due to tension within tethered collagen gels is a useful tool for generating anisotropic tissues, but requires an optimal balance between cell density, matrix concentration and time to be achieved for each specific cell type. The aim of this study was to develop an assay system based on contraction of free-floating cellular gels in 96-well plates that could be used to investigate cell-matrix interactions and to establish optimal parameters for subsequent self-alignment of cells in tethered gels. Using C6 glioma cells, the relationship between contraction and alignment was established, with 60-80% contraction in the 96-well plate assay corresponding to alignment throughout tethered gels made using the same parameters. The assay system was used to investigate the effect of C6 cell density, collagen concentration and time. It was also used to show that blocking α1 integrin reduced the contraction and self-alignment of these cells, whereas blocking α2 integrin had little effect. The approach was validated by using primary astrocytes in the assay system under culture conditions that modified their ability to contract collagen gels. This detailed investigation describes a robust assay for optimising cellular self-alignment and provides a useful reference framework for future development of self-aligned artificial tissue.

Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture.

Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.

Brucellosis in childhood.

Brucellosis has always been an unusual disease in children and, concomitant with the control of the disease in domestic animals, reports have become sparse. The pediatrician, therefore, may not be aware of the protean clinical manifestations of childhood brucellosis. In 1973, nine cases occurred during a three-month period in El Paso, Texas. All cases were marked by spiking fevers and lethargy of four days to four weeks in duration. Tender hepatomegaly or splenomegaly was striking in seven patients. Other characteristics included epistaxis, arthralgia, myalgia, and weight loss. Leukopenia and leukemoid reaction were found in five patients. All of the patients tested had elevated liver enzymes. Febrile agglutinins were invaluable in screening for an early clue to diagnosis. When Brucella abortus antigen agglutinated serum from patients with a positive screen in dilutions greater than 1:320, a presumptive diagnosis of brucellosis was made. Brucella was isolated from the blood or bone marrow in seven patients and the time of incubation proved crucial for successful recovery. Bacterial blood cultures are usually discarded at ten days of age, as were cultures from the only two patients from whom the organism was not recovered. All of the cultures incubated for 12 to 15 days grew B. melitensis, an unusual causative species in the United States. However, several patients admitted eating cheese from the State of Chihuahua, Mexico, made from unpasteurized goat's milk, the presumed source of the infection. Within one to three days, all patients responded dramatically to antibiotics; tetracycline was given orally for 21 days and streptomycin intramuscularly for 14 days. Pediatricians caring for patients in areas where consumption of unpasteurized milk products is likely would do well to consider brucellosis in a child with obscure fever or toxic hepatosplenomegaly.