Human adipocyte differentiation and characterization in a perfusion-based cell culture device.

Adipocytes have gained significant attention recently, because they are not only functioning as energy storage but also as endocrine cells. Adipocytes secret various signaling molecules, including adiponectin, MCP-1, and IL-6, termed collectively as "adipokines". Adipokines regulate glucose metabolism, thereby play an important role in obesity, diabetes type 2, and other metabolic disorders. Conventionally, to study the secretory function, adipocytes are cultured in vitro in static conditions. However, static culturing condition falls short of mimicking the interstitial fluid flows in living systems. Here, we developed a perfusion device which allows dynamic culture of adipocytes under constant and mild flow using a double-layered fluidic structure. Adipocytes were cultured in the bottom layer while the culture media were constantly flown in the upper layer and perfused through a porous membrane that separate the two chambers. The porous membrane between the two chambers physically separates the cells from the flow stream while maintain a fluidic connection by diffusion. This setting not only provides continuous nutrient supply to adipocytes but also maintains a steady and mild shear stress on the cell membrane. It was found the perfusion-based culture conditions promoted faster growth of primary preadipocytes and stimulated greater adipogenesis compared to static culture condition. Adipocytes cultured under perfusion systems produced more MCP-1 and IL-6, but less adiponectin. When stimulated with TNF-α, adipocytes expressed higher level of MCP-1 and IL-6, but lower level of adiponectin. No significant glucose uptake regulation was observed after treating the adipocytes with insulin in both static and perfusion-based culture. Our results demonstrate that perfusion-base culture has played a role in the adipocyte function particularly the secretion of adipokines. More future studies are required to unveil the mechanisms behind perfusion's impact on adipocytes.

On-chip immune cell activation and subsequent time-resolved magnetic bead-based cytokine detection.

Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of triple-layered microfluidic chambers was fabricated to enable simultaneous cell culture under perfusion flow and detection of the induced cytokines at multiple time-points. Each culture chamber comprises three fluidic compartments which are dedicated to, cell culture, perfusion and immunoassay. The three compartments are separated by porous membranes, which allow the diffusion of fresh nutrient from the perfusion compartment into the cell culture compartment and cytokines secretion from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture and without minimizing benefit gain from perfusion. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The cytokines released from differential stimuli were quantified in situ in non-differentiated U937 monocytes and differentiated macrophages.

Anoctamin 6 is an essential component of the outwardly rectifying chloride channel.

Outwardly rectifying chloride channels (ORCC, ICOR) of intermediate single-channel conductance of around 50 pS, are ubiquitously expressed, but have remained a mystery since their description more than 25 y ago. These channels have been shown to be activated on membrane excision and depolarization of the membrane voltage and by cAMP in the presence of the cystic fibrosis transmembrane conductance regulator. We show that anoctamin 6 (Ano6), a member of the recently identified family of putative Cl(-) channels, is the crucial component of ORCC single-channel and whole-cell currents in airway epithelial cells and Jurkat T lymphocytes. Cystic fibrosis transmembrane conductance regulator augmented ORCC produced by Ano6 in A549 airway epithelial cells. Ano6 is activated during membrane depolarization or apoptosis of Jurkat T lymphocytes and epithelial cells, and is inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid, 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid, or AO1. Ano6 belongs to the basic equipment of any cell type, including colonic surface epithelial cells. It forms the essential component of ORCC and seems to have a role for cell shrinkage and programmed cell death.

Development of a cellulose-based 96-well plate vertical flow pull-down assay.

The abundance and low production cost of biomaterial cellulose paper have attracted attention for many applications. Point-of-care (PoC) diagnostic tests have been successfully developed using patterned cellulose paper. Although PoC diagnostic tests are rapid and simple to perform, their sample processing throughput is limited, allowing for only one sample to be evaluated at a time, which restricts potential applications. Thus, it was appealing to expand cellulose-based PoC tests to high-throughput versions to increase their applicability. Here, we present the development of a high-throughput cellulose-based 96-well plate vertical flow pull-down assay that can process 96 tests, is easy to prepare, and can be customized for different detection targets. The device has two key features: (i) patterned cellulose paper for 96 tests that do not require pre-immobilization of capturing reagents, and (ii) reusable sturdy housing. We believe that a variety of applications, including laboratory testing, population surveillance tests, and sizable clinical trials for diagnostic tests, can benefit from the adoption of this cellulose-based 96-well plate assay.

F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis.

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.

Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A.

TMEM16A (anoctamin 1, Ano1), a member of a family of 10 homologous proteins, has been shown to form an essential component of Ca(2+)-activated Cl(-) channels. TMEM16A-null mice exhibit severe defects in epithelial transport along with tracheomalacia and death within 1 mo after birth. Despite its outstanding physiological significance, the mechanisms for activation of TMEM16A remain obscure. TMEM16A is activated on increase in intracellular Ca(2+), but it is unclear whether Ca(2+) binds directly to the channel or whether additional components are required. We demonstrate that TMEM16A is strictly membrane localized and requires cytoskeletal interactions to be fully activated. Despite the need for cytosolic ATP for full activation, phosphorylation by protein kinases is not required. In contrast, the Ca(2+) binding protein calmodulin appears indispensable and interacts physically with TMEM16A. Openers of small- and intermediate-conductance Ca(2+)-activated potassium channels known to interact with calmodulin, such as 1-EBIO, DCEBIO, or riluzole, also activated TMEM16A. These results reinforce the use of these compounds for activation of electrolyte secretion in diseases such as cystic fibrosis.

Expression and function of epithelial anoctamins.

Endogenous Ca(2+)-activated Cl(-) currents (CaCCs) are abundant and present in very different cell types. Very good evidence has been provided that endogenous CaCC is produced by anoctamin 1 (Ano1) and Ano2. Insight into the physiological role of anoctamins has been provided for Ano1, Ano2 and Ano6; however, the physiological role of the other seven members of the anoctamin family remains obscure. Anoctamins 1 and 2 may operate as individual Ca(2+)-sensitive channel proteins or may require accessory subunits for complete function. We find that overexpressed Ano1 has properties resembling all those of endogenous CaCCs, although with some noticeable biophysical and regulatory differences when compared with endogenous channels. Apart from Ano1 and Ano2, expression of Ano6 also produces a Cl(-) conductance. Depending on the cellular background, Ano6 currents may have variable properties. Anoctamins 1 and 6 are frequent in epithelial cells, often coexpressed together with Ano8, Ano9 and Ano10. Most available data on anoctamins were obtained from mouse tissues and from cultured cells, which may not be representative of native human tissues.

Generation of Thermally Stable Affinity Pairs for Sensitive, Specific Immunoassays.

Many point-of-care diagnostic tests rely on a pair of monoclonal antibodies that bind to two distinct epitopes of a molecule of interest. This protocol describes the identification and generation of such affinity pairs based on an easily produced small protein scaffold rcSso7d which can substitute monoclonal antibodies. These strong binding variants are identified from a large yeast display library. The approach described can be significantly faster than antibody generation and epitope binning, yielding affinity pairs synthesized in common bacterial protein synthesis strains, enabling the rapid generation of novel diagnostic tools.

Development and translation of a paper-based top readout vertical flow assay for SARS-CoV-2 surveillance.

Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.

Finger stick blood test to assess postvaccination SARS-CoV-2 neutralizing antibody response against variants.

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population.