Cell Catcher: A New Method to Extract and Preserve Live Renal Cells from Urine

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Introduction A proportion of urinary tract cells are shed into urine by using normal physiological processes. These include epithelial cells from kidney tubules, podocytes, as well as immune and bladder cells.1 Urine-derived cells offer advantages over biopsies because they are easily obtained repeatedly without pain or discomfort. Furthermore, they have various research uses, including modeling genetic kidney disorders,2 generating stem cells,3 and drug screening.4,5 Despite these advantages, the full potential of urine-derived cells is not being realized. One key issue is inconsistencies between methods to initiate cell cultures from urine. Healthy adult urine contains between 2.5 and 7.5 cells/100 ml, which can proliferate in culture, yielding millions of cells within 2–4 weeks.6,7 However, the success rate of initiating and expanding cells from urine is variable, ranging from 10% to 73%.3,8–10 In addition, the cell population obtained is heterogeneous, containing both differentiated and undifferentiated cells,11 complicating the interpretation of studies. These inconsistencies in yields and cell identity are likely due to methodological differences, including culture conditions. Currently, urinary cells are isolated within 4 hours of sample collection, using two-step centrifugation, requiring a laboratory in close proximity to the collection site. This makes the process logistically challenging and extends the time cells are exposed to urine affecting cell viability.1 We hypothesized that immediate processing through filtration will improve the yield of cultured cells obtained from urine compared with centrifugation. This is because filtration minimizes urine exposure time, decreases processing times, and reduces mechanical stress on cells. To test this, we developed a filtration-based Cell Catcher device for processing urine at clinical sites and directly compared its efficiency with centrifugation. Methods Urine was collected from consented patients at the Royal Free Hospital, St Thomas Hospital, and Great Ormond Street Hospital, London (Ethical approval references: 05/Q0508/6, 08/H0713/82). First, samples from 18 tubulopathy patients (Supplemental Table 1) were equally split by volume for paired analyses. One was processed by the Cell Catcher device (Figure 1A) within 30 minutes of collection at the clinic and the other transported on ice to a laboratory and centrifuged within 4 hours (Figure 1B). In a second study, we assessed samples from tubulopathy patients (n=18), adult and pediatric patients with Bardet–Biedl syndrome (BBS, n=15), and healthy controls (n=11) (Supplemental Table 2). In these 44 individuals, the whole sample volume was processed either by Cell Catcher or centrifugation (Figure 1C). Finally, a further six samples from tubulopathy patients were split and processed by either the Cell Catcher or centrifugation (Supplemental Table 3), and cell viability and phenotype were assessed before culture.Figure 1: Cell Catcher clinical validation study. (A) Cell Catcher diagram. The hub of the Cell Catcher has detachable lids and houses a membrane. It connects to a detachable funnel for urine samples to be processed by gravity. After filtration, media are added for the cells to be preserved during transport, inside the hub. Prototypes were produced using Polyjet 3D printing (University College London, B-made 3D printing, Bartlett School of Architecture). (B) Split-sample study design. Eighteen samples were collected from patients with renal tubulopathies; each sample was split into two equal parts—Cell Catcher group and centrifugation group. Sample fractions in the Cell Catcher group were processed on site within 30 minutes of collection and stored at room temperature for up to 4 hours during transportation to the laboratory, where they were plated. Sample fractions in the centrifugation group were stored at 4°C and transported to the laboratory on ice within 4 hours to be centrifuged and plated. (C) Whole-sample study design. Forty-four samples were collected from patients with renal tubulopathies, BBS, and healthy adults, and whole volume was processed either by Cell Catcher or centrifugation. (D) Culturing outcomes, split-sample study. Cells from sample fractions processed by either a Cell Catcher or centrifugation were seeded, cultured, and assigned to the following categories: no clusters (by 2 weeks), clusters (day 6), and contamination (within the first day). Distribution of the three culturing outcomes for each experimental condition is shown. (E) Split-sample cell yield differences between Cell Catcher and centrifugation fractions. Cell clusters (>10 cells) were quantified on day 6 after plating, by two researchers independently. Average numbers of the two counts are plotted for each of the 11 samples in the paired study, where clusters formed in at least one of the fractions (Wilcoxon nonparametric paired t test, n=11, P = 0.001). (F) Culturing outcomes, whole-sample study. Cells from samples processed by either the Cell Catcher or centrifugation were seeded, cultured, and assigned to the following categories: no clusters (by 2 weeks), clusters (day 6), and contamination (within the first day). Distribution of the three culturing outcomes for each experimental condition is shown. (G) Whole-sample cell yield differences between Cell Catcher and centrifugation-processed samples. Cluster counts in samples processed by either Cell Catcher (n=18) or centrifugation (n=16), after two outliers that were identified in each group were removed. Mann–Whitney test, P = 0.0013. Median+interquartile range is plotted. BBS, Bardet–Biedl syndrome.Cell Catcher is a patent-pending (Encelo Laboratories Ltd.) custom-built device manufactured using 3D printing that houses a polyethersulfone 5-µm membrane (Sterlitech). Gravity-fed filtration can be achieved for samples <100 ml with low specific gravity (SG, 1.005–1.015) or <25 ml of high-SG samples (1.020–1.030) with all steps performed at room temperature. After filtration, the outer funnel was removed, a bottom lid attached to the hub, and 12 ml of medium (DMEM High Glucose/F12 [1:1], 1% penicillin/streptomycin, 1% amphotericin B, 10% FBS, human EGF, insulin, hydrocortisone, transferrin, triiodothyronine, epinephrine, bovine pituitary extract, and adenine) added to the cells contained in the upper portion of the hub. Centrifuged samples were processed as per published protocols.12 In samples assessed for viability before culture, cell pellets were resuspended in 10 μl of medium, stained using trypan blue and counted by an automated machine (BioRad) to measure total cell count and percentage of live cells/ml of urine collected. The cells in three of these samples processed by the Cell Catcher were used for flow cytometry analysis. Cell pellets were resuspended in block (PBS with 5% mouse and rat serum) and incubated with allophycocyanin anti-human CD13 (Biolegend) and phycoerythrin anti-human podoplanin (Biolegend) antibodies as panepithelial and podocyte markers, respectively. Cells were washed, resuspended in PBS containing 2% FBS, and analyzed on FACSymphony A5 (BD Biosciences) with the population gated to exclude debris and dead cells. All other cell samples were plated in a 12-well tissue culture dish and incubated at 37°C and 5% CO2. Half of the media was replaced with fresh once daily until day 3 and replaced completely every 2 days thereafter. Cells were passaged at around 80% confluence. Cultures were monitored daily and assigned to the following categories: contaminated (defined as bacterial infection within 1 day of culture, despite an initial negative result on urinalysis strips), formed cell clusters, and no clusters. Where cell clusters (>10 cells) formed, the number was counted on day 6 of culture independently by two investigators. RNA was extracted at first passage of the cells using the RNeasy Plus Mini kit (Qiagen); 500 ng was used to synthesize cDNA using the iScript gDNA Clear

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