Membranes for biohybrid liver support systems--investigations on hepatocyte attachment,morphology and growth

The biological properties of four different membranes were studied regarding their possible application in biohybrid liver support systems. Two of them, one made of polyetherimide (PEI), and a second based on polyacrylonitrile-N-vinylpyrollidone co-polymer (P(AN-NVP)), were recently developed in our lab and studied for the first time. Together with pure polyacrylonitrile (PAN) membranes, the three preparations were characterised as ultra-filtration membranes. Their ability to support cell attachment, morphology, proliferation and function of human hepatoblastoma C3A cells was studied. The role of surface morphology for the interaction with hepatocytes was highlighted using a commercial, moderately wettable polyvinylidendifluoride (PVDF) membrane with micro-filtration properties. Comparative investigations showed strongest interaction of C3A cells with PAN membranes, as the focal adhesion contacts were more expressed and cell growth was also high. However, the functional activity in terms of albumin synthesis was reduced. Very similar results were obtained with the most hydrophobic PEI membrane. In contrast, the most hydrophilic membrane P(AN-NVP) was found to provoke stronger homotypic adhesion (E-cadherin expression) of C3A cells and less substratum attachment (focal adhesions), but enhanced albumin secretion. However, proliferation of C3A cells was lowered. Micro-porous PVDF membrane showed very good initial attachment, but the resulting cell material and cell-cell interaction were relatively poor developed. Among four membranes tested, PEI seems to be the most attractive membrane for biohybrid liver devices, as it provides good surface properties for hepatocytes interaction, but in addition it is highly thermostable, which would permit steam sterilisation. No simple relationship, however, between the wettability of the membranes and their ability to support hepatocyte adhesion and function was found in this study.     

Nonradiometric detection of cytotoxicity of teleost nonspecific cytotoxic cells

The development of a sensitive, rapid and reliable nonradiometric cytotoxicity assay would significantly facilitate studies of teleost nonspecific cytotoxic cells. Such an assay would not require handling and disposal of radionuclides and it would not depend on secondary enzyme or colorimetric determinations. The requirements for this assay would consist of a one-step binding protocol which could detect early target cell membrane lesions and at very low effector:target cell ratios. In this chapter, we have developed a flow cytometry based cytotoxicity assay utilizing the uptake of propidium iodide (PI) into cells containing damaged (i.e. permeabilized) cell membranes. The basis of detection of cellular damage depended on flow discrimination of targets from effector cells by establishing 'scatter' gates from these mixtures. Teleost (catfish) anterior kidney NCC were mixed with human transformed targets (IM-9 and HL-60 cells) at effector:target cell ratios of 1, 5 and 10 and PI uptake was determined at 3 and 14 hours post-incubation. Percent specific uptake (PSU) was calculated by determining total binding capacity (TBC) (i.e. uptake of PI by cold acetone permeabilized target cells) and spontaneous binding capacity (SBC) (i.e. PI uptake by target cells incubated in media w/o effectors). This was represented by the formula PSU = [T – SBC/TBC – SBC] × 100 where T is the PI uptake of targets following addition of effector cells. Using this technique, NCC initiated target cell permeabilization as early as 30 minutes co-incubation (25:1 E:T ratio) and damaged membranes were detected in mixtures containing as few as 1:1 effector:target cell ratios. At 5:1 E:T ratios, greater than 50 PSU was determined following 14 hours co-incubation. Using these criteria, a new and sensitive cytotoxicity assay has been developed to determine NCC activity.     

New modified polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes

There has been growing interest in innovative materials with physico-chemical properties that provide improved blood/cell compatibility. We propose new polymeric membranes made of modified polyetheretherketone (PEEK-WC) as materials with potential for use in biohybrid devices. PEEK-WC exhibits high chemical, thermal stability and mechanical resistance. Owing to its lack of crystallinity this polymer can be used for preparing membranes with cheap and flexible methods. We compared the properties of PEEK-WC membranes to polyurethane membranes prepared using the same phase inverse technique and commercial membranes. The physico-chemical properties of the membranes were characterised by contact angle measurements. The different parameters acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) of the surface free energy were calculated according to Good-van Oss's model. We evaluated the cytocompatibility of PEEK-WC membranes by culturing hepatocytes isolated from rat liver. Cell adhesion and metabolic behaviour in terms of ammonia elimination, urea synthesis and protein synthesis were evaluated during the first days of culture. Liver cells adhered and formed three-dimensional aggregates on the most tested membranes. PEEK-WC membranes promoted hepatocyte adhesion most effectively. Urea synthesis, ammonia elimination and protein synthesis improved significantly when cells adhered to PEEK-WC membrane. The considerable metabolic activities of cells cultured on this membrane confirmed the good structural and physico-chemical properties of the PEEK-WC membrane that could be a promising biomaterial for cell culture in biohybrid devices.     

Morphological studies on the culture of kidney epithelial cells in a fiber-in-fiber bioreactor design with hollow fiber membranes

A hollow fiber-in-fiber-based bioreactor system was tested for the applicability to host kidney epithelial cells as a model system for a bioartificial kidney. Hollow fibers were prepared from polyacrylonitrile (PAN), polysulfone-polyvinylpyrollidinone (PVP) blend (PSU) and poly(acrylonitrile-N-vinylpyrollidinone) copolymer P(AN-NVP). Hollow fibers with smaller and larger diameters were prepared so that the smaller fitted into the larger, with a distance of 50-100 microm in between. The following material combinations as outer and inner fiber were applied: PAN-PAN; PSU-PSU, PSU-P(AN-NVP). Madin-Darby kidney epithelial cells (MDCK) were seeded in the interfiber space and cultured for a period up to 14 days. Light, scanning, and transmission electron microscopy were used to follow the adhesion and growth of cells, and to characterize their morphology. As a result, we found that MDCK cells were able to grow in the interfiber space in mono- and multilayers without signs of systemic degeneration. Comparison of the different materials showed that PAN and P(AN-NVP) provided the best growth conditions, indicated by a tight attachment of cells on hollow fiber membrane, and subsequent proliferation and development of structural elements of normal epithelia, such as tight junctions and microvilli. In conclusion, the fiber-in-fiber design seems to be an interesting system for the construction of a bioartificial kidney.     

Membranes for biohybrid liver support: the behaviour of C3A hepatoblastoma cells is dependent on the composition of acrylonitrile copolymers

Co-polymers based on acrylonitrile, N-vinylpyrrolidone, aminoethylmethacrylate and sodium methallylsulfonate were used to prepare flat membranes by phase inversion. The surface properties of membranes were characterised by water contact angle measurements, atomic force microscopy and X-ray photoelectron spectroscopy (XPS). Membrane permeability was estimated by porosity measurements with water as test liquid. Human C3A hepatoblastoma cells were plated on these materials. Cell-material interaction was characterised by overall cell morphology, formation of focal adhesion contacts and intercellular junctions. Furthermore, cell proliferation was measured and compared with the functional activity of cells as indicated by 7-ethoxycoumarin-O-deethylation. More hydrophilic materials reduced spreading of cells, formation of focal adhesion and subsequent proliferation while homotypic cell adhesion was facilitated in correlation with stronger expressions of intercellular junctions and improved functional activity. In contrast, membranes with stronger adhesivity enhanced cell proliferation but reduced the functional activity of cells. It was concluded that the co-polymerisation of acrylonitrile with hydrophilic co-monomers, such as N-vinylpyrrolidone, could be used to tailor membrane materials for the application in biohybrid liver support systems.     

Novel Polymer Blends for the Preparation of Membranes for Biohybrid Liver Systems

It was found previously that membranes based on co-polymers of acrylonitrile (AN) and 2-acrylamido-2-methyl-propansulfonic acid (AMPS) greatly stimulated the functionality and survival of primary hepatocytes. In those studies, however, the pure AN–AMPS co-polymer had poor membrane-forming properties, resulting in quite dense rubber-like membranes. Hence, membranes with required permeability and optimal biocompatibility were obtained by blending the AN–AMPS co-polymer with poly(acrylonitrile) homopolymer (PAN). The amount of PAN (P) and AN–AMPS (A) in the blend was varied from pure PAN (P/A-100/0) over P/A-75/25 and P/A-50/50 to pure AN–AMPS co-polymer (P/A-0/100). A gradual decrease of molecular cut-off of membranes with increase of AMPS concentration was found, which allows tailoring membrane permeability as necessary. C3A hepatoblastoma cells were applied as a widely accepted cellular model for assessment of hepatocyte behaviour by attachment, viability, growth and metabolic activity. It was found that the blend P/A-50/50, which possessed an optimal permeability for biohybrid liver systems, supported also the attachment, growth and function of C3A cells in terms of fibronectin synthesis and P-450 isoenzyme activity. Hence, blend membranes based on a one to one mixture of PAN and AN–AMPS combine sufficient permeability with the desired cellular compatibility for application in bioreactors for liver replacement.     

Surface modification of the polymers present in a polysulfone hollow fiber hemodialyser by covalent binding of heparin or endothelial cell surface heparan sulfate: flow characteristics and platelet adhesion

The present study addresses the problem of simultaneous surface modification of various polymers, i.e. polysulfone (PSU), polycarbonate (PC), and polyurethane (PU), which constitute the Ultraflux AV 600 S hollow fibre hemodialyser. An investigation was first made into six different chemical routes aimed at introducing carboxyl groups onto the surface of PSU, PC, and PU model polymers to which heparin (HE) or endothelial cell surface heparan sulfate (ESHS) was covalently bound via the reaction of residual amino groups and a coupling reagent. Carboxyl groups were introduced using three specific reactions based on their nucleophilic or electrophilic introduction into aromatic repeating units of the polymers and three non-specific carboxylation reactions, i.e. UV, heat or redoxactivation via nitrene or radical species. Concentrations of 1-20 nmol COOH groups per cm(-2) led to HE or ESHS surface concentrations corresponding to one or several layers. Two nonspecific carboxylation reactions followed by HE- or ESHS-coupling provided the lowest change in membrane pore structure according to cut off, clearance (urea, phosphate, maltose), ultrafiltration, and diafiltration assessments. In some cases the introduction of excess negatively-charged carboxyl groups and HE improved the flux properties of the modified membranes. The various methods were applied to the dialysis module. Platelet adhesion was not observed in the case of the ESHS-coating of PSU membrane at shear rates of 1050 s(-1), whereas HE and subendothelial matrix showed 56 and 100% coverage, respectively, under similar conditions. The coating of PSU or of other high-flux membranes by ESHS appears a promising method for improving membrane properties and to generate biocompatibility characteristics similar to those of natural blood vessels, i.e. inertness to platelet adhesion and no level effects for complement and intrinsic coagulation cascade activation. The ESHS coating may be used without anticoagulants.     

Surface modification of the polymers present in a polysulfone hollow fiber hemodialyser by covalent binding of heparin or endothelial cell surface heparan sulfate: Flow characteristics and platelet adhesion

The present study addresses the problem of simultaneous surface modification of various polymers, i.e. polysulfone (PSU), polycarbonate (PC), and polyurethane (PU), which constitute the Ultraflux AV 600 S® hollow fibre hemodialyser. An investigation was first made into six different chemical routes aimed at introducing carboxyl groups onto the surface of PSU, PC, and PU model polymers to which heparin (HE) or endothelial cell surface heparan sulfate (ESHS) was covalently bound via the reaction of residual amino groups and a coupling reagent. Carboxyl groups were introduced using three specific reactions based on their nucleophilic or electrophilic introduction into aromatic repeating units of the polymers and three non-specific carboxylation reactions, i.e. UV, heat or redoxactivation via nitrene or radical species. Concentrations of 1-20 nmol COOH groups per cm^-2 led to HE or ESHS surface concentrations corresponding to one or several layers. Two nonspecific carboxylation reactions followed by HE- or ESHS-coupling provided the lowest change in membrane pore structure according to cut off, clearance (urea, phosphate, maltose), ultrafiltration, and diafiltration assessments. In some cases the introduction of excess negatively-charged carboxyl groups and HE improved the flux properties of the modified membranes. The various methods were applied to the dialysis module. Platelet adhesion was not observed in the case of the ESHS-coating of PSU membrane at shear rates of 1050 s^-1, whereas HE and subendothelial matrix showed 56 and 100% coverage, respectively, under similar conditions. The coating of PSU or of other highflux membranes by ESHS appears a promising method for improving membrane properties and to generate biocompatibility characteristics similar to those of natural blood vessels, i.e. inertness to platelet adhesion and no level effects for complement and intrinsic coagulation cascade activation. The ESHS coating may be used without anticoagulants.     

Porous and single-skinned polyethersulfone membranes support the growth of HepG2 cells: a potential biomaterial for bioartificial liver systems

In this study, we evaluated a porous and single-layer skin polyethersulfone (PES) membrane as a material for use in hybrid bioartificial liver support systems. The PES membrane has been characterized as a single-layer skin structure, with a rough porous surface. Specifically, we studied the ability of the human hepatoblastoma cell lines (HepG2) to adhere, grow, and spread on the PES membrane. Furthermore, we examined albumin secretion, low-density lipoprotein uptake, and CYP450 activity of HepG2 cells that grew on the membrane. HepG2 cells readily adhered onto the outer surfaces of PES membranes. Over time, HepG2 cells proliferated actively, and confluent monolayer of cells covered the available surface area of the membrane, eventually forming cell clusters and three-dimensional aggregates. Furthermore, HepG2 cells grown on PES membranes maintained highly specific functions, including uptake capability, biosynthesis and biotransformation. These results indicate that PES membranes are potential substrates for the growth of human liver cells and may be useful in the construction of hollow fiber bioreactors. Porous and single-layer skin PES membranes and HepG2 cells may be potential biomaterials for the development of biohybrid liver devices.     

Evaluation of adhesion, proliferation, and functional differentiation of dermal fibroblasts on glycosaminoglycan-coated polysulfone membranes

It may be hypothesized that wound healing will benefit from polymer membranes coated with extracellular matrix macromolecules. Here we describe the behavior of dermal fibroblasts on polysulfone (PSU) membranes, and PSU membranes covered with chitosan (Ch), chondroitin sulfate (CS), or hyaluronan, using an additional intermediary ionic charge modification with poly-(acrylonitrile-co-methallyl sulfonate) (AN69), which allows binding of the polysaccharidic macromolecules. Cell adhesion, proliferation, cell signaling, and collagen gene expression were investigated. Ch and CS were found unable to support cell adhesion and proliferation. In contrast, both PSU and hyaluronic acid-coated PSU membranes appeared as suitable materials to culture fibroblasts and support their matrix synthesis capacity. Moreover, they induce type III collagen expression in addition to type I, suggesting that they promote a fetal-like environment that could be beneficial for wound healing.     

In vitro activation of human complement by polyacrilonitrile dialyzer membrane assayed by complement binding to bystander red cells.

Quantitation of complement activation by polyacrilonitrile (PAN) dialyzer membrane is complicated by the high adsorptive capacity of the membrane for fluid phase anaphylotoxins. Assays for these anaphylotoxins, therefore, underestimate the degree of complement activation produced by this membrane. Alternative methods of measuring in vitro complement activation by the PAN and Cuprophan membranes were explored by incubating normal human erythrocytes with the membranes in the presence of serum. This led to deposition of C3d on these "innocent bystander" red cells, and provided an independent parameter for measuring complement activation. The PAN membrane caused significantly more C3d deposition on red cells, and thus more complement activation than Cuprophan. The possible significance of complement activation by PAN membrane, in consideration of its property of binding the resultant anaphylotoxins, is discussed.     

Surface characteristics of acrylic modified polysulfone membranes improves renal proximal tubule cell adhesion and spreading

Current polyvinylpyrrolidone-modified polysulfone (PVP-PSU) membranes in haemodialysers do not facilitate the attachment and proliferation of renal proximal tubule cells (RPTCs). For bioartificial kidney (BAK) development expensive extracellular matrices are employed to ensure the PVP-PSU membranes can serve as a substrate for RPTCs. In this study we modified PSU using an acrylic monomer (am-PSU) and polymerization using ultraviolet irradiation. We demonstrated that on adjusting the PSU or acrylic content of the membranes the wettability and surface chemistry were altered, and this affected the amount of fibronectin (Fn) that was adsorbed onto the membranes. Using an integrin blocking assay we ascertained that Fn is an important extracellular matrix component that mediates RPTC attachment. The amount of Fn adsorbed also led to different bioresponses of RPTCs, which were evaluated using attachment and proliferation assays and qualitative quantification of vinculin, focal adhesion kinase, zonula occludens and Na(+)/K(+) ATPase. Our optimized membrane, am-PSU1 (21.4% C-O groups, 19.1% PVP-PSU; contact angle 71.5-80.80, PVP-PSU: 52.4-67.50), supports a confluent monolayer of RPTCs and prevents creatinine and inulin diffusion from the apical to the basal side, meeting the requirements for application in BAKs. However, further in vivo evaluation to assess the full functionality of RPTCs on am-PSU1 is required.     

Polyethersulfone Block Copolymers for Membrane Applications

Polyarylethersulfones (polysulfone: PSU, polyethersulfone: PESU) belong to the group of established membrane materials and are mainly used for production of ultra‐ and microfiltration membranes. They offer several advantageous properties like hydrolysis and chemical resistance, mechanical strength and bio‐compatibility. A general drawback of these polymers compared to the more polar membrane materials cellulosetriacetate or polyacrylonitrile is the hydrophobic nature of the polyarylethersulfone which can be quantified by the contact angle versus water. An improved synthetic pathway to segmented block copolymers based on polyarylethersulfones and various aliphatic polyethers is presented as well as the characterization of the obtained block copolymers and their use for membrane preparation.     

Nanofiber membrane of graphene oxide/polyacrylonitrile with highly efficient antibacterial activity

Abstract

Infection from bacterial resistance to antibiotics has given rise to a grave threat to human health in the world. It is vital to developing highly efficient antibacterial materials that are safe and biocompatible with humans and without bacterial resistance. In this study, nanofiber membranes of graphene oxide/polyacrylonitrile (GO/PAN) with highly efficient antibacterial activity were fabricated via electrospinning technique. As the spindle-knot structure of membranes formed by the addition of GO sheets increased, the hydrophilicity and surface roughness increased. The antibacterial test indicated that antibacterial ratios of 3GO/PAN membranes against Escherichia coli and Staphylococcus aureus were 98.5% and 99.6%, respectively after contracting 24?h, with highly efficient antibacterial activity. Furthermore, the E. coli cell structures of adhered to the GO/PAN nanofiber surface changed significantly shrunk and deformed, and the number of S. aureus cell were obviously less contrast than the pure PAN. The main antibacterial mechanism was GO of spindle-knot in nanofiber membranes produced ROS destroyed the physiological activities of the bacteria lead to death. The fabricated GO/PAN nanofiber membrane of this study is promising to develop to a kind of novel antibacterial materials.     

Artificial cells as bioreactive biomaterials

Artificial cells can have the same dimensions as biological cells. They can enclose and retain a large variety of bioreactants. Artificial cell membranes can have the required permeability characteristics to allow the rapid equilibration of external molecules to be acted on by the enclosed bioreactants. Polymer, protein, protein-lipid, polymer-lipid, lipid or other materials can form artificial cell membranes. It is possible to vary the membrane thickness and permeability characteristics over a wide range. Many different forms of bioreactive artificial cells are available. This article contains only a few examples. This includes its applications in (1) red blood cell substitutes; (2) immobilisation of enzymes, multienzymes with cofactor recycling, cell cultures and other biotechnological applications; (3) hemoperfusion in kidney failure, poisoning, removal of aluminum and iron; (4) use in liver failure; (5) other applications in microencapsulation.     

Epithelial mesenchymal interactions,the ECM and limb development

It has been long since recognized that cellular interactions are not always direct, i.e. they do not always take place between cells contacting each other, or between cells that emit soluble factors and other cells, which respond to it. In contrast, cross-talk between cells is frequently based on signals attached to the extracellular matrix (ECM). Thus besides proximate cell-to-cell contact, certain interactions are mediated by the ECM in a sequence: cell-to-matrix, matrix-to-cell. ECM-mediated interactions may take place within a group or sheet of cells or across adjacent cell sheets. A modified mat-like ECM, the basement membrane, separates adjacent cell sheets and mediates their interactions. Since cell sheets separated by basement membranes are an elementary feature of metazoan histology, interactions with the basement membrane have considerable importance. Recently accumulated evidence emphasizes the importance of ECM-mediated interactions. It is becoming increasingly evident that the ECM functions not only as an architectural component, but it is involved also in signal transduction. This evidence derives from four main sources: from the structure of receptor-ligand complexes, from Drosophila and C elegans genetics, from cell biological observations and from the analysis of mammalian development. In this review, I will touch upon recent evidence, illustrated by examples of FGF signalling in vertebrate limb development. Although the involvement of matrix components is not yet proven for all cases directly, the strength of multiple indications suggests that a better understanding of ECM-mediated interactions will shed new light on cell differentiation.     

Glomerular albumin filtration through podocyte cell body in puromycin aminonucleoside nephrotic rat

It is an a priori concept that protein molecules including albumin are filtrated through the slit membrane between the foot processes of podocytes. However, foot processes are effaced and the number of slit membranes is reduced in nephrotic syndrome, suggesting another pathway of albumin filtration through the foot process cell body. Thus, we investigated the pathway of gold-and fluorescein isothiocyanate (FITC)-labeled albumin filtration in the puromycin aminonucleoside (PAN) model of nephrotic syndrome in the rat. PAN rats at day 7 with established nephrotic proteinuria were injected with 8-nm gold-labeled albumin and FITC-labeled albumin through the jugular vein followed by kidney fixation at 10 or 30 min. Goldlabeled albumin was accumulated in the paramesangial area and in the endosomes of glomerular endothelial cells of both control and PAN rats by electron microscopy. On the other hand, FITC-labeled albumin was detected between foot processes in the control but more in the podocyte cell body in the PAN rat. In conclusion, albumin will be filtrated through the decreased numbers of slit diaphragms; however, albumin can be also taken up in the podocyte, the mesangium, and the glomerular endothelium, suggesting that there might be other routes of glomerular albumin clearance in nephrotic syndrome.     

Intercellular contacts in portions of early atherosclerotic lesions of the human aorta]

The ultrastructure of human thoracic aortas collected at autopsy from 16 trauma victims aged from 3 to 40 years was investigated. Intercellular contacts were found in all aortas examined. The contacting cells localized predominantly in aortic areas predisposed to atherosclerosis, i. e. in areas situated around the ostia of the intercostal arteries. Most of contacting cells were monocytes--macrophages (Mn/Mph) which interacted with foam cell and intimal smooth muscle cells (SMCs). Only occasional contacts were formed by lymphocytes and endothelial cells. All intimal contacting SMCs were of the synthetic phenotype, i. e. they did not contain myofilaments and had well developed endoplasmic reticulum and Golgi apparatus. In thickened aortic intima the contacting cells were seen with the signs of destruction. The data obtained suggest that the contacts between intimal cells may be the consequence of the immune reaction directed to the transfer of information, cell cytolysis and utilization of cell debris.     

Hemocompatibility and anaphylatoxin formation of protein-immobilizing polyacrylonitrile hemodialysis membrane

Plasma proteins were covalently immobilized onto polyacrylonitrile (PAN) membrane to evaluate the hemocompatibility and anaphylatoxin formation. This is used as a model to study the effect of protein-adsorption on the blood-contacting response of hemodializing membranes. The proteins used were either platelet-adhesion-promoting collagen (COL) or platelet-adhesion-inhibiting human serum albumin (HSA). The microstructure and characterization of the protein-immobilizing PAN membranes were evaluated by Coomassie dye assay, atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle measurement. PAN-HSA membrane improved not only hemocompatibility including less platelet adhesion, longer blood coagulation times, and higher thrombin inactivity level, but also induced lower complement activation. On the other hand, PAN-COL membrane exhibited blood incompatibility, although induced less increase of C3, C4 antigens of serum. Overall results of this study demonstrated that the immobilization of HSA onto the surface of PAN membrane would be beneficial to improve the hemocompatibility and to reduce the anaphylatoxin formation during hemodialysis.     

P300 amplitude reduction is associated with early‐onset and late‐onset pathological substance use in a prospectively studied cohort of 14‐year‐old adolescents

P3 amplitude reduction (P3AR) is associated with risk for adolescent‐onset pathological substance use (PSU). In this longitudinal study, data from over 1,100 adolescent twins were used to examine P3AR in relation to early adolescent onset PSU (i.e., by age 14), late adolescent onset PSU (i.e., ages 14–18), misuse of different classes of substances (PSU‐nicotine, PSU‐alcohol, PSU‐illicit), degree of PSU comorbidity, and gender differences. P3 amplitude was recorded at age 14 from two midline electrodes during a visual oddball paradigm. PSU was defined as meeting criteria for any symptom of a substance use disorder assessed using semistructured clinical interviews. P3AR was associated with degree of drug class comorbidity, early adolescent onset PSU for all three substance classes, and late adolescent onset PSU for alcohol and illicit PSU. Gender differences in P3AR were not statistically significant. These findings provide further evidence that P3AR indexes a nonspecific diathesis for adolescent‐onset PSU.