Visualizing medium and biodistribution in complex cell culture bioreactors using in vivo imaging.

There is a dearth of technology and methods to aid process characterization, control and scale-up of complex culture platforms that provide niche micro-environments for some stem cell-based products. We have demonstrated a novel use of 3d in vivo imaging systems to visualize medium flow and cell distribution within a complex culture platform (hollow fiber bioreactor) to aid characterization of potential spatial heterogeneity and identify potential routes of bioreactor failure or sources of variability. This can then aid process characterization and control of such systems with a view to scale-up. Two potential sources of variation were observed with multiple bioreactors repeatedly imaged using two different imaging systems: shortcutting of medium between adjacent inlet and outlet ports with the potential to create medium gradients within the bioreactor, and localization of bioluminescent murine 4T1-luc2 cells upon inoculation with the potential to create variable seeding densities at different points within the cell growth chamber. The ability of the imaging technique to identify these key operational bioreactor characteristics demonstrates an emerging technique in troubleshooting and engineering optimization of bioreactor performance.

A novel automated bioreactor for scalable process optimisation of haematopoietic stem cell culture.

Proliferation and differentiation of haematopoietic stem cells (HSCs) from umbilical cord blood at large scale will potentially underpin production of a number of therapeutic cellular products in development, including erythrocytes and platelets. However, to achieve production processes that are scalable and optimised for cost and quality, scaled down development platforms that can define process parameter tolerances and consequent manufacturing controls are essential. We have demonstrated the potential of a new, automated, 24×15 mL replicate suspension bioreactor system, with online monitoring and control, to develop an HSC proliferation and differentiation process for erythroid committed cells (CD71(+), CD235a(+)). Cell proliferation was relatively robust to cell density and oxygen levels and reached up to 6 population doublings over 10 days. The maximum suspension culture density for a 48 h total media exchange protocol was established to be in the order of 10(7)cells/mL. This system will be valuable for the further HSC suspension culture cost reduction and optimisation necessary before the application of conventional stirred tank technology to scaled manufacture of HSC derived products.

Candida albicans hyphal invasion: thigmotropism or chemotropism?

Hyphae of the human pathogenic fungus Candida albicans exhibit thigmotropic behaviour in vitro, in common with phytopathogenic and saprotrophic fungi. An examination of the literature on C. albicans hyphal penetration of epithelial and endothelial membranes does not support the premise that hyphal thigmotropism plays a major role in tissue invasion. Further experimentation is now required to assess thigmotropic behaviour on host membranes and vaginal epithelial cells are suggested as a test model. It is proposed that while thigmotropism may and invasion of tissue invaginations, chemotropism can explain C. albicans hyphal invasion patterns of both endothelium and epithelium.

A gene sequence expressed only in undifferentiated EC, EK cells and testes.

A clone, EC1, has been isolated from a cDNA library prepared from 4-day embryoid bodies formed by suspension culture of PSMB EC cells. This clone has been used to screen a variety of RNA sources including adult tissues, embryonal carcinoma (EC), and endoderm cell lines. A 3-kb poly(A)+ RNA species was found to be present only in undifferentiated EC cells and adult mouse testes. This species was significantly reduced in testes of W/Wv mice compared with wild-type at this locus. Germ cells and their progeny are therefore implicated as the source of the RNA in testes. Hybrid-selected RNA from PSMB could be translated in vitro into a 35-kd protein, but no translatable message was evident in either PYS-2 (parietal), or PSA5-E (visceral) endoderm cell lines. DNA sequencing of the EC1 insert revealed that it is 744 bp in length, the 3' 460 bp of which are in open reading frame. Comparison with known sequences have shown no significant homology. EC1 subclones in M13 have been used to generate single-stranded probes for hybridisation to RNA in situ in tissue sections. Hybridisation of the strand complementary to RNA produces a signal limited to the central regions of embryoid bodies formed on suspension culture of embryo-derived EK cells coinciding with the presence of undifferentiated cells. Probing of a mouse genomic library and Southern blots of liver DNA with EC1 reveals that the gene is present as a single copy.

Thermocouple thermometry in microwave fields.

The causes of some of the artefacts and hazards resulting from the use of thermocouples in a 434 MHz microwave field have been investigated. The factors influencing their magnitudes are discussed.

Applications of xeroradiography in dentistry-a review.

Xeroradiography is the technique in which electrostatically charged plates sensitive to X-rays are used in diagnostic radiology in place of conventional film. There has however been anxiety that radiation dosage for xeroradiography may be at unacceptably high levels. James et al., however, in 1973 showed that by increasing the kilovoltage to at least 120 the exposure could be reduced by 60 per cent. Using higher kV lateral oblique jaw and lateral and anteroposterior skull xeroradiographs have been produced with lower radiation exposure than conventional film. Bony detail is much more sharply delineated on xeroradiographs and soft tissues are visible on the same picture without use of a grid or wedge filter. These features are of obvious advantage in cephalometrics and orthognathic surgery. Panoramic techniques are potentially the most useful way of applying xeroradiography. The combination of full jaw coverage with the sharp definition only possible at present with intra-oral radiographs would provide more information for the dentist, save time for the radiographer and reduce the dose to the patient. Excellent results have been obtained with autopsy specimens on machines which develop 90kVp, but optimal exposure for a normal adult requires a panoramic X-ray machine development 120 kVp. Xeroradiography has the advantage, therefore, of providing more detail of diagnostic value with lower radiation exposure to the patient. The process requires no silver, which is in increasingly short supply.

The advantages of xeroradiography for panoramic examination of the jaws and teeth.

Xeroradiography is an electrostatic imaging method which was significant advantages over conventional radiographic techniques. This article introduces the use of xeroradiography for panoramic examination of the jaws. The resultant images not only contain superior local contrast and detail when compared to conventional panoramic radiographs but also correlate well with histopathologic changes. The investigation indicates that xeroradiography is an important new diagnostic tool for detection of disease of the jaws and teeth.

Radiation exposure to the patient in xeroradiography.

The paper presents measurements of the radiation exposure required in xeroradiography of the breast and of the extremities. The nature of the radiation hazard and the most appropriate quantity by which to assess it are discussed, and it is shown that for a number of radiological procedures xeroradiographs can be taken for about the same radiation exposure to the patient as that required by conventional film techniques.

The detection and significance of calcifications in the breast: a radiological and pathological study.

The radiological detection of calcification is compared using xeroradiography, non-screen film and a film-screen combination. The "threshold" values of the smallest detectable size of calcification, under simulated clinical conditions, are found to be approximately 100 mum for xeroradiography and 400 mum for both the film techniques in this study. The incidence of calcification seen on the preoperative mammograms of patients with carcinoma of the breast is 48-5 per cent. Further calcification revealed by histological examination raises the overall incidence of calcification in mammary carcinomas to 63 per cent. The incidence on preoperative mammograms in benign breast disease is 20 per cent. The radiological features of calcification occurring in malignant and benign breast lesions are recorded, and no definitive distinguishing features are established. The histological appearance of calcification in malignant and benign breast disease is discussed.