RPTEC Not Growing Like They Should? This is What You Might Be Doing Wrong
Renal proximal tubule epithelial cells (RPTEC) play an essential role within the kidney, functioning to transport waste products into the lumen while simultaneously reabsorbing water, salt, and organic solutes from the glomerular filtrate. Characterized by a polarized morphology, these highly specialized cells have been widely studied by researchers wishing to better understand renal drug reabsorption, elimination, and toxicity. Unfortunately, RPTEC culture can be challenging, with in vitro models often failing to accurately represent the in vivo situation. By culturing RPTEC appropriately, in a manner which implements polarization, researchers can more reliably evaluate novel drugs.
A quick recap on the physiology reabsorption and secretion
Selecting the right RPTEC line
Researchers working with RPTEC use both primary and immortalized cells. An advantage of primary cells is that they typically express most proximal tubule transporters, providing more physiologically relevant data, however, they have only a finite lifetime and often exhibit high batch variation. Immortalized cells can be grown indefinitely in culture, yet they usually lose key transporters and characteristics over time, casting doubt upon results. A further disadvantage of immortalized RPTEC is that many commercially available lines do not demonstrate contact inhibition, a key morphological property. This means that they over-grow in culture and are no longer suitable for experimentation.
At MIMETAS, we use human RPTEC modified with CompoZr® zinc finger nuclease (ZFN) technology to extend cell proliferation. These exhibit the combined benefits of primary and immortalized cultures, making them ideally suited to a wide variety of kidney cell-based assays.
Choose a culture method to implement polarization
One of the biggest mistakes a researcher can make when culturing RPTEC is to grow them as a monolayer on a plastic surface. In vivo, RPTEC are polarized, with the apical side located at the lumen of the proximal tubules where it is exposed to direct flow, and the basolateral side facing the blood and functioning to provide the cells with fresh nutrients. It is impossible to reproduce this situation within a cell culture flask.
Although Transwell® permeable supports have been used in attempts to better mimic in vivo conditions, these systems provide only limited information since they are unsuitable for high-resolution kinetic measurements and image-based readouts. Supporting up to 96 tissue models concurrently within a microfluidic 3D cell culture plate, OrganoPlate® technology facilitates RPTEC polarization for unprecedented quantification and imaging.
OrganoPlate® better represents the in vivo situation
Within OrganoPlate®, RPTEC are continuously perfused by medium as they grow against a freestanding extracellular matrix (ECM) layer. Permeable for nutrients and compounds applied to the basolateral side of the tubule, the ECM layer is stiff enough to allow the cells to grow as a tight barrier. This enables researchers to perform barrier integrity assays, and to study the transport function of the proximal tubule, applications for which a tight barrier is critical.
A perfusion channel on the other side of the ECM allows apical access to the RPTEC tubule, however a unique feature of OrganoPlate® is that additional culture lanes can easily be added to increase the complexity of tissue models. For example, using the perfusion channel to co-culture an endothelial tubule, a configuration permitting perfusion of both tubules concurrently, these interdependent yet non-homogeneous tissues can easily be studied in synchrony.