A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum β-lactamase-positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration.

Objective Studying the biology of the human placenta represents a major experimental challenge. Although conventional cell culture techniques have been used to study different types of placenta-derived cells, current in vitro models have limitations in recapitulating organ-specific structure and key physiological functions of the placenta. Here we demonstrate that it is possible to leverage microfluidic and microfabrication technologies to develop a microengineered biomimetic model that replicates the architecture and function of the placenta. Materials and methods A “Placenta-on-a-Chip” microdevice was created by using a set of soft elastomer-based microfabrication techniques known as soft lithography. This microsystem consisted of two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane. To reproduce the placental barrier in this model, human trophoblasts (JEG-3) and human umbilical vein endothelial cells (HUVECs) were seeded onto the opposite sides of the ECM membrane and cultured under dynamic flow conditions to form confluent epithelial and endothelial layers in close apposition. We tested the physiological function of the microengineered placental barrier by measuring glucose transport across the trophoblast-endothelial interface over time. The permeability of the barrier study was analyzed and compared to that obtained from acellular devices and additional control groups that contained epithelial or endothelial layers alone. Results Our microfluidic cell culture system provided a tightly controlled fluidic environment conducive to the proliferation and maintenance of JEG-3 trophoblasts and HUVECs on the ECM scaffold. Prolonged culture in this model produced confluent cellular monolayers on the intervening membrane that together formed the placental barrier. This in vivo-like microarchitecture was also critical for creating a physiologically relevant effective barrier to glucose transport. Quantitative investigation of barrier function was conducted by calculating permeability coefficients and metabolic rates in varying conditions of barrier structure. The rates of glucose transport and metabolism were consistent with previously reported in vivo observations. Conclusion The “Placenta-on-a-Chip” microdevice described herein provides new opportunities to simulate and analyze critical physiological responses of the placental barrier. This system may be used to address the major limitations of existing placenta model systems and serve to enable research platforms for reproductive biology and medicine.

No abstract

No abstract

Human epidermal cells grew and differentiated in vitro, provided that the pH of the culture medium was at 5.6-5.8, the seeding density was optimal (-2.5 X 105 cells per cm2), and the incubation temperature was maintained at 35-37°C. Under these conditions, epidermal cells from many different skin locations grew to confluency within 15-20 days and formed multi-layered sheets whose differentiated structure resembled that of the full depth of skin epidermis. Cell proliferation and differentiation did not require a feeder layer, a collagen substrate, a high concentration of fetal bovine serum, or added hormones. The sheets of differentiated epidermal cells could be dissociated from the plastic surfaces of the tissue culture flasks. The use of such cultured cells for wound dressing is proposed.The apparent dependence of human epidermal cell growth and differentiation in vitro on the presence of a mouse 3T3 cell feeder layer (1) or collagenized substrates (2, 3) has led to the supposition that epidermal cell growth and differentiation in vivo may depend on dermal cell products. Epidermal cell growth has also been observed in the absence of such supports, but then the medium had to be supplemented by hormone preparations such as pituitary extracts and a high concentration (20%) of fetal bovine serum (4). Thus, it has been difficult to study the role of cell-to-cell interactions, and the influences of substances such as hormones, chalones, etc. on the process of epidermal cell differentiation in vitro because of the obligatory presence of nonepidermal components in the system. We now present evidence tht epidermal growth in vitro demands neither dermal elements nor special nutrients, provided conditions of pH, seeding density, and incubation temperature have been optimized. MATERIALS AND METHODSTissue Culture. More than 200 human skin specimens from different skin locations (scalp, face, neck, arms, breast, foreskin, legs, and trunk) as well as skin shavings from burn victims and cadavers (4-6 hr after death) have been successfully cultivated. Both full and split-thickness skin have been used. Skin samples were freed from fatty tissue and washed in minimal essential medium (GIBCO) with Earle's salts, containing, per ml, 1000 units of penicillin, 1 mg of streptomycin, and 2.5 ug of Fungizone for 30 min, followed by two washes, of 10 min each, in the same medium. Two additional washes 10 min each were in minimal essential medium with 1/10th the concentration of antibiotics. Discs of tissue were cut from the epidermal side of each specimen with sharp curved scissors, including as little dermis as possible. After washing in 0.02% EDTA (Sigma), the pieces were transferred to 0.25% trypsin (1:250, Difco) at 4°C for 12-15 hr. Subsequent to this incubation, the cut pieces were transferred to a fresh dish and the epidermis of each piece was detached from its dermis with fine forceps. Isolated epidermal samples were pooled in a trypsin/EDTA solution (5) [8 g of NaCl, 0.4 g of KCI, 1 g of dextrose, 0.58 g of NaHCO3, 0.5 g ...