This post is meant to serve as an abbreviated version of an internal White Paper and is provided as an update to our efforts here at DiscoveryBioMed, Inc. (DBM, Inc.) to continue to optimize and develop human ADPKD kidney cell platforms. We are ‘learning by doing’ as always, and we wanted to present some new findings and current status of products in development.
A Mixed Cell Population Is Key to “Cystogenesis Functionality”
One confounding issue in the practice of isolating, growing, genotyping and characterizing single cyst-derived primary cultures and multicystic/microcystic tissue-derived primary cultures is that not all of the primary cultures from individual kidneys form cysts or are capable of cystogenesis. This is puzzling in light of the fact that they all derive from the same human ADPKD donor kidney tissue. Having said that, with each donor, we do have primary cultures that indeed form cysts. During the characterization of all primary cultures from a given donor in 3D Biogels, we also seed some cells from each culture on TC plastic so as to image and assess whether there is a single cystic epithelial cell type in the culture or if there is more than one cell type in the culture. In the context of this work, there is a uniform phenotype that a mixed cell population appears necessary in a given huADPKD primary culture to form cysts.
Not only do we observe routinely both small and large cystic epithelial cells in these ‘cystogenesis capable’ cultures, but we also observe a small number of fibroblasts as well. There are typically not enough fibroblasts in these cultures to overgrow and overwhelm these cultures and this is tested for in quality control to ensure consistency between cultures. Interestingly, a common practice in epithelial cell culture (cystic or non-cystic) is to eliminate fibroblasts from the cultures to generate a ‘pure’ population of epithelial cells. DBM believes this may be short sighted. For example, we have separated ‘pure’ populations of epithelial cells and fibroblasts from a given primary culture. However, before we separated the cell types, the culture formed cysts in 3D, whereas after separation, cystogenesis failed to occur, suggesting an indispensable role of fibroblasts in this process. It is also possible that these fibroblasts are actually progenitor cells and one academic investigator in Europe believes that this may be the case. Taken together, there is more than one type of cell in these cultures and multiple types of epithelial cell and we and others continue to define them going forward.
Current Iteration of the 3D Cystogenesis Bioassays
DBM can perform three different types of cystogenesis assays with coded and blinded client LTAs. Cystogenesis occurs in our DBM RenalCyte media only and does not require stimuli at significant concentrations. More recently, we have begun to test different stimuli with forskolin and arginine vasopressin accelerating cyst formation and increasing cyst number. Growth factors do not seem to accelerate the system, suggesting that the cells themselves may produce and secrete necessary growth factors endogenously in the Biogels.
First, the “Cyst Attack” assay tests a candidate therapeutic LTA on cysts that have already formed and become an ideal size to determine whether the LTA stops further cyst enlargement, shrinks the cysts, causes them to collapse into a spheroid, or rupture altogether. Imaging is performed daily over time in these custom assays where cyst number per well and cyst size can be measured. Overall viability is measured via 3D CellTiterGLO cell quantification (based on cytosolic ATP from live cells) at the conclusion of the experiment.
Second, the “Cyst Prevention” assay tests a candidate therapeutic LTA on Biogels where cysts have just begun to form, so as to determine whether they attenuate or prevent cyst formation versus untreated and vehicle-treated controls.
Third, in “Cyst Suppression” assays, one can treat from the initial seeding of the cells into the Biogels (versus controls) and prevent the appearance of any cysts from time 0 or Day 0 for a prescribed period of time. Similar endpoints to the “Cyst Attack” assay can be captured.
Recently, DBM procured a BioTek Cytation 5 high content imaging and light-based high-throughput screening imaging system so as to image the huADPKD cells growing in 3D Biogels in 96-well and 384-well microtiter plates. This effort seeks to ‘miniaturize’ the assay from the current 48-well plate format. DBM has been successful in observing cystogenesis from key primary and immortalized huADPKD cell cultures in 96-well full area well plates, 96-well half area well plates and in 384-well plates. This effort seeks to increase the throughput of the assay and also allows for implementation of automated robotic light-based bioassays and high-content imaging in the future for our clients. The use of fluorescent dyes are currently being investigated to enhance image acquisition and quality.
Current Status of Immortalized huADPKD Cell Cultures
In 2017, DBM began by challenging cystogenesis competent cells from three of our six donor cultures, two of which have mutations in PKD1 and one which has a mutation in PKD2. From these cultures, we derived immortal cell cultures using both SV40T and hTERT. These immortalized mixed cultures are still being characterized, but at early passage form 3D cysts in Biogels similar to their primary progenitors. We are continuing to passage these cells to observe phenotype and if the hTERT immortalized cultures maintain growth and phenotype, we will select them with neomycin. We are also employing additional immortalization strategies.
Please enquire to learn more about either our primary or immortal ADPKD cell cultures and assays.
Continue Reading »