Schematic representation of the continuous media perfusion setup.

<p>A syringe pump is used to pump media through gas-permeable tubing to adjust gaseous tension levels before entering the culture device.</p

Modelling of flow conditions in the microfluidic chip.

<p>(<b>a</b>) represents the velocity field at half the height of the inlet channel. (<b>b</b>) represents the velocity field 15 um above the culture plane (ACP). (<b>c</b>) shows velocity profiles at x₀ along the z-axis.</p

Quantification of hESC colony size.

<p>Due to the time required to image larger culture areas, only the central area of the control dish, which contained the majority of colonies, was imaged. The numbers of distinct colonies shrinks as nearby colonies grow into each other.</p

Monitoring a hESC colony in the microfabricated culture device during the course of an experiment.

<p>The same colony is shown after (a) 1 day static culture, (b) 1 day perfused culture and (c) 2 days perfused culture. The columns show, from left to right, the raw phase contrast image taken with a 4× objective, an overlay of the automated detection using the image processing algorithm, and the detected area. The scale bars are 500 µm.</p

Design of the microfabricated culture device.

<p>(<b>a</b>) Exploded view showing all parts of the modular microfabricated culture device. (<b>b</b>) Schematic representation of a longitudinal section of the interconnect assembly, showing compression of the PDMS chip around the inlet/outlet ports (dashed rectangle), by the interconnect. (<b>c</b>) Top view of the microfluidic chip with dashed lines showing the footprints of the lid and interconnect bosses. (<b>d</b>) Cross-sectional view showing the two PDMS layers of the microfluidic chip. The lower ‘spacer’ layer elevates the flow equalisation barriers of the top layer and thus reduces the hydrodynamic shear exposure for the cells.</p

Staining of hESC colonies following perfusion culture.

<p>Representative images of the feeder cells and hESC colonies in the culture device after 2 days of continuous perfusion culture. Each row shows the phase contrast images (<b>a</b>, <b>e</b>) of the feeder-attached hESC colonies and the corresponding results from DAPI (<b>b</b>, <b>f</b>,) and pluripotency marker staining for Oct-4 (<b>c</b>), Tra-1-81 (<b>d</b>) and SSEA-3 (<b>g</b>). All images were taken with a 20× objective, scale bar is 200 µm.</p

Co-cultured hESCs in microfabricated culture device and control dish.

<p>Representative phase contrast images of iMEF feeder cells and individual hESC colonies cultured in the microfabricated culture device (<b>a-c</b>) and in the control dishes (<b>d-f</b>). The same two colonies are shown at each of three time points; after 1 day of static culture (a, d), after 1 day of the perfused culture (b, e), and at the end of the 2 days of perfused culture (c, f). All images were taken with a 4× objective, scale bar is 500 µm.</p

Photograph of the assembled modular culture device with the re-sealable lid attached.

<p>Photograph of the assembled modular culture device with the re-sealable lid attached.</p

Analysis of Gene Expression Profiles of Microdissected Cell Populations Indicates that Testicular Carcinoma In situ Is an Arrested Gonocyte

Testicular germ cell cancers in young adult men derive from a precursor lesion called carcinoma in situ (CIS) of the testis. CIS cells were suggested to arise from primordial germ cells or gonocytes. However, direct studies on purified samples of CIS cells are lacking. To overcome this problem, we performed laser microdissection of CIS cells. Highly enriched cell populations were obtained and subjected to gene expression analysis. The expression profile of CIS cells was compared to microdissected gonocytes, oogonia and cultured embryonic stem cells (ESCs) with and without genomic abberations. Three samples of each tissue type were used for the analyses. Unique expression patterns for these developmentally very related cell types revealed that CIS cells were very similar to gonocytes as only five genes distinguished these two cell types. We did not find indications that CIS was derived from a meiotic cell and the similarity to ESCs was modest compared to gonocytes. Thus we provide new evidence that the molecular phenotype of CIS cells is similar to that of gonocytes. Our data are in line with the idea that CIS cells may be gonocytes that survived in the postnatal testis. We speculate that disturbed development of somatic cells in the fetal testis may play a role in allowing undifferentiated cells to survive in the postnatal testes. The further development of CIS into invasive germ cell tumors may depend on signals from their post-pubertal niche of somatic cells, including hormones and growth factors from Leydig and Sertoli cells