Low-cost microphysiological systems: Feasibility study of a tape-based barrier-on-chip system for small intestine modeling

We see affordability as a key challenge in making organs-on-chips accessible to a wider range of users, particularly outside the highest-resource environments. Here, we present an approach to barrier-on-a-chip fabrication based on double-sided pressure-sensitive adhesive tape and off-the-shelf polycarbonate. Besides a low materials cost, common also to PDMS or thermoplastics, it requires minimal (€ 100) investment in laboratory equipment, yet at the same time is suitable for upscaling to industrial roll-to-roll manufacture. We evaluate our microhpysiological system with an epithelial (C2BBe1) barrier model of the small intestine, studying the biological effects of permeable support pore size, as well as stimulation with a common food compound (chili pepper-derived capsaicinoids). The cells form tight and continuous barrier layers inside our systems, with comparable permeability but superior epithelial polarization compared to Transwell culture, in line with other perfused microphysiological models. Permeable support pore size is shown to weakly impact barrier layer integrity as well as the metabolic cell profile. Capsaicinoid response proves distinct between culture systems, but we show that impacted metabolic pathways are partly conserved, and that cytoskeletal changes align with previous studies. Overall, our tape-based microphysiolgical system proves to be a robust and reproducible approach to studying physiological barriers, in spite of its low cost.

Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3)

The collecting duct is the primary site of urinary ammonia secretion; the current study determines whether apical ammonia transport in the mouse inner medullary collecting duct cell (mIMCD-3) occurs via nonionic diffusion or a transporter-mediated process and, if the latter, presents the characteristics of this apical ammonia transport. We used confluent cells on permeable support membranes and examined apical uptake of the ammonia analog [14C]methylammonia ([14C]MA). mIMCD-3 cells exhibited both diffusive and saturable, transporter-mediated, nondiffusive apical [14C]MA transport. Transporter-mediated [14C]MA uptake had a Kmof 7.0 ± 1.5 mM and was competitively inhibited by ammonia with a Kiof 4.3 ± 2.0 mM. Transport activity was stimulated by both intracellular acidification and extracellular alkalinization, and it was unaltered by changes in membrane voltage, thereby functionally identifying an apical, electroneutral NH4+/H+exchange activity. Transport was bidirectional, consistent with a role in ammonia secretion. In addition, transport was not altered by Na+or K+removal, not inhibited by luminal K+, and not mediated by apical H+-K+-ATPase, Na+-K+-ATPase, or Na+/H+exchange. Finally, mIMCD-3 cells express the recently identified ammonia transporter family member Rh C glycoprotein (RhCG) at its apical membrane. These studies indicate that the renal collecting duct cell mIMCD-3 has a novel apical, electroneutral Na+- and K+-independent NH4+/H+exchange activity, possibly mediated by RhCG, that is likely to mediate important components of collecting duct ammonia secretion.

Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3)

The renal collecting duct is the primary site for the ammonia secretion necessary for acid-base homeostasis. Recent studies have identified the presence of putative ammonia transporters in the collecting duct, but whether the collecting duct has transporter-mediated ammonia transport is unknown. The purpose of this study was to examine basolateral ammonia transport in the mouse collecting duct cell (mIMCD-3). To examine mIMCD-3 basolateral ammonia transport, we used cells grown to confluence on permeable support membranes and quantified basolateral uptake of the radiolabeled ammonia analog [14C]methylammonia ([14C]MA). mIMCD-3 cell basolateral MA transport exhibited both diffusive and transporter-mediated components. Transporter-mediated uptake exhibited a Kmfor MA of 4.6 ± 0.2 mM, exceeded diffusive uptake at MA concentrations below 7.0 ± 1.8 mM, and was competitively inhibited by ammonia with a Kiof 2.1 ± 0.6 mM. Transporter-mediated uptake was not altered by inhibitors of Na+-K+-ATPase, Na+-K+-2Cl−cotransporter, K+channels or KCC proteins, by excess potassium, by extracellular sodium or potassium removal or by varying membrane potential, suggesting the presence of a novel, electroneutral ammonia-MA transport mechanism. Increasing the outwardly directed transmembrane H+gradient increased transport activity by increasing Vmax. Finally, mIMCD-3 cells express mRNA and protein for the putative ammonia transporter Rh B-glycoprotein (RhBG), and they exhibit basolateral RhBG immunoreactivity. We conclude that mIMCD-3 cells express a basolateral electroneutral NH4+/H+exchange activity that may be mediated by RhBG.

Deactivation of TSH receptor signaling in filter-cultured pig thyroid epithelial cells

Thyrotropin [thyroid-stimulating hormone (TSH)] receptor on-off signaling was studied in polarized monolayers of pig thyrocytes cultured on permeable support. Transepithelial resistance (R) and potential difference (PD) were used as parameters to monitor the effect of altered TSH concentrations on vectorial electrolyte transport. TSH induced rapid but long-lasting changes in R (decrease) and PD (increase) that were cAMP-dependent and related to enhanced transcellular conductance of sodium and chloride. Withdrawal of TSH from cultures prestimulated with TSH (0.1 mU/ml) for 48 h resulted in restitution of R to control level within 30 min. Such deactivation was markedly accelerated by mild trypsinization, which degraded receptor-bound ligand without affecting TSH receptor responsiveness or ion transporting capacity. Small alterations in the TSH concentration (0.01–0.1 mU/ml) were followed almost instantaneously by adjustments of R. In contrast, the reversal of R after acute TSH stimulation (30 min) and subsequent TSH washout was delayed for several hours independently of cell surface trypsinization. The observations indicate that, during continuous exposure to physiological concentrations, TSH exerts a close minute-to-minute surveillance of thyroid function and the rate-limiting step of deactivation is the dissociation of ligand from the TSH receptor at the cell surface. TSH-deprived cells briefly exposed to TSH are refractory to rapid deactivation, probably because of altered metabolism downstream of TSH receptor signal transduction.

Regulation of sodium transport in M-1 cells

The M-1 cell line, derived from the mouse cortical collecting duct (CCD), is being used as a mammalian model of the CCD to study Na+ transport. The present studies aimed to further define the role of various hormones in affecting Na+ transport in M-1 cells grown in defined media. M-1 cells on permeable support, in serum-free media, developed amiloride-sensitive current 4–5 days after seeding. As expected for the involvement of epithelial Na+ channels, α-, β-, and γ-subunits of the epithelial Na+ channel were identified by RT-PCR. Either dexamethasone (Dex, 10–100 nM) or aldosterone (Aldo, 10−6–10−7M) for 24 h stimulated transport. Cells grown in the presence of Aldo and Dex had higher transport than with Dex alone. Spironolactone added to Dex media decreased transport. The acute effects of hormones reported to inhibit Na+ transport in CCD were also examined. Epidermal growth factor, phorbol esters, and increased intracellular Ca2+ with thapsigargin did not alter transport. Arginine vasopressin caused a transient increase in transport (probably Cl− secretion), which was not amiloride sensitive. Also, the protease inhibitor aprotinin decreased Na+ transport; in aprotinin-treated cells, trypsin stimulated transport. This study demonstrates that adrenal steroids (Dex > Aldo) stimulate Na+ transport in M-1 cells. At least part of this response may represent activation of mineralocorticoid receptors based on an additive effect of Dex and Aldo, as well as inhibition by spironolactone. Responses to immediate-acting hormones is limited. However, an endogenous protease activity, which activates Na+ transport, is present in these cells.

ATP decreases acutely and reversibly transport through the paracellular pathway in human cervical cells

We studied the effect of ATP on transepithelial transport through the paracellular pathway in human cervical cells. Transepithelial conductance and transepithelial permeability (determined from the measurements of unidirectional flux of inert molecules) were measured in Caski cells grown on permeable support. Transepithelial conductance was 55.9 +/- 17.7 mS/cm2 and permeability was 12.5 +/- 2.7 x 10(-6) cm/s for a 0.51-kDa probe. Addition of ATP to the medium decreased acutely and reversibly the conductance and the permeability to probes between 0.18 and 10 kDa by 23-31% in a dose-related fashion; the 50% effective concentration was 1 microM, with a maximal effect at 5-10 microM extracellular ATP. The ATP effect was observed regardless of the pressure gradient across the epithelium. These results indicate that extracellular ATP in micromolar concentrations decreases acutely and reversibly the permeability through the paracellular pathway in cervical cells, possibly by affecting the permeability of the tight junctions and the resistance of the intercellular space. On the basis of these data, we speculate that ATP may play a role in the regulation of solutes and fluid transport across the cervical epithelium in vivo.