Enterobacteriaceae resistant to tetracycline and sulfonamide antibiotics, and the taxonomic composition of the microbiota by sequencing the 16S rRNA genes of one kit per nest. Changing the surrounding microbes of the kits can hinder the transmission of antibiotic resistance genes from one generation to the next, but the three communities widely differed in their ability to orient gut microbes and in their impact on antibiotic resistance genes. The most efficient delivery of the microbial community reduced the proportion of resistant Enterobacteria from 93 to 9%, decreased the relative abundance of eight antibiotic resistance genes, and changed the gut microbes of the kits at weaning. The least efficient did not reduce any ARG or modify the bacterial community. In addition, adding fecal pellets was more efficient than the oral inoculation of the anaerobic suspension derived from these fecal pellets. However, we were unable to predict the outcome of the exclusion from the data of the donor does (species composition and abundance of antibiotic resistance genes). In conclusion, we revealed major differences between microbial communities regarding their ability to exclude antibiotic resistance genes, but more work is needed to understand the components leading to the successful exclusion of antibiotic resistance genes from the gut. As a consequence, studies about the impact of competitive exclusion should use several microbial communities in order to draw general conclusions.Reducing antibiotic use is a necessary step toward less antibiotic resistance in livestock, but many antibiotic resistance genes can persist for years, even in an antibiotic-free environment. In this study, we investigated the potential of three fecal complex microbial communities from antibiotic-naive does to drive the microbiota of kits from antibiotic-exposed dams and outcompete bacteria-carrying antibiotic-resistant genes. The fecal complex microbial communities were either orally delivered or simply added as fresh fecal pellets in four to five nests that were kept clean from maternal feces. Additionally, four nests were cleaned for the maternal feces and five nests were handled according to the common farm practice  as controls. At weaning, we measured the relative abundance of 26 antibiotic resistance genes, the proportion of  Microbes exposed to antibiotics in livestock may develop resistance mechanisms, including a variety of targets such as efflux systems, drug modifiers or changes in the target’s configuration . As a maCampylobacter spontaneously evolved a resistance against macrolides and was not outcompeted by its sensitive kin in an antibiotic-free environment  that were acquired from a French rabbit farm for which the antibiotic history was available. The dams were selected so that they all received diclazuril (coccidiostat), tilmicosin (macrolide), sulfadimethoxine (sulfonamide), oxytetracycline and trimethoprim. They also received a subcutaneous injection of 23 mg/kg of sulfadimethoxine and 5 mg/kg trimethoprim twice a day for three days and 100 mg/kg oxytetracycline by a single subcutaneous injection 25 days before their due date. They arrived in the animal facility of the French National Institute for Agricultural Research (INRA) 18 days before their parturition. In contrast, the three non-gravid “donor” does  producing the competitive microbial communities were from farms that have banned the use of antibiotics for at least 5 years.2. The content was then homogenized using vigorous shaking.The fecal suspensions tested for competitive exclusion were prepared less than 2 h before their oral administration: for each donor doe, five fecal pellets (approx. 5 g) were added to a bottle containing 5 g of 1-mm glass beads and 40 mL 0.9% NaCl. After the bottle was sealed with a septum, oxygen was purged through a 0.1-mm needle via five cycles of pumping at −700 mbar and filling with 500 mbar NEnterobacteriaceae, and 10 colonies per sample were stored at −80°C in order to later test their resistance to antibiotics. At Day 3, 17, and 35, fresh fecal pellets from maternal and the three donor does were recovered and placed in dry ice. A 2-g subsample was homogenized with a bagmixer  for isolation and storage of Enterobacteriaceae, as previously described for kit fecal samples. At Day 35, a fecal pellet from each doe was immediately stored at −80°C for DNA extraction.Gravid dams were fed with a standard diet  until weaning. They were raised in cages measuring 100 × 42 × 60 cm that included 24 × 42 cm nests that could be closed by a sliding door. Suckling was allowed from 8 to 8:30 am for the first 21 days. From Day 1 to 21, dams had no access to their kits other than during the suckling window, which is common practice until Day 7 for meat rabbit farming in France. The litters were equilibrated to 6–8 kits per litter at Day 1. The 37 dams that had already given birth (Day 0) were divided into eight groups. The kits in the control group were raised as usual; the kits from the “No Feces” control group  were raised as usual except that we removed all maternal fecal pellets after suckling up to Day 21. For the kits of the six treated groups, we also removed the maternal fecal pellets after suckling but we put them in contact with the fecal microbes of a “donor” Doe  until Day 29. Each donor doe was kept individually in cages located in different rooms for the whole duration of the experiment. The groups I41, I43, and I44  received the microbial suspensions prepared as described above by letting them suck 100 μl from a syringe every second day between 10 and 11 am from Day 2 to 29. Gloves and syringes were changed between each cage to avoid cross-contamination. For the groups P41, P43, and P44 , we put five fresh fecal pellets from the relevant donor doe in the nest containing the kits. At Day 21, the nests were opened and kits could freely roam in the whole cage and interact with the dam until Day 36. At Day 35, one kit per cage was anesthetized via an intramuscular injection of 80 mg ketamine/kg  followed by an intracardiac injection of 182 mg pentobarbital/kg , causing death. The experiments were conducted under the agreement number APAFIS-2018021215568441 for animal experimentation from the French Ministry of Agriculture (Ethics Committee C2EA-86). The fecal pellets were immediately recovered from the digestive tract and a 100-mg subsample was placed in dry ice and then stored at −80°C for DNA extraction. Another 500-mg subsample was mixed with 3 mL of sterile phosphate-buffered peptone water containing 30% w/w glycerol and immediately mixed with a pestle by hand. The resulting homogenate was plated on MacConkey medium to isolate Enterobacteriaceae isolates collected from the does or the kits was tested by plating a 24-h pure culture onto Mueller-Hilton agar supplemented with 16 μg/mL tetracycline or 128 μg/mL sulfamethoxazole according to the ECOFF E. coli epidemiological cut-off values defined by Eucast . Each test included a resistant and sensitive control strain to confirm that the antibiotic was active and that the incubation conditions allowed growth. The average percentage of antibiotic-resistant Enterobacteriaceae isolates was calculated for each lactating doe (maximum of 30 isolates with 10 isolates per sampling day) and for their nest (maximum of 30 isolates with 10 isolates for one kit per nest). When the number of isolates was too low to calculate an average percentage (n < 10 for the doe or the nest) and/or when the average percentage of antibiotic-resistant Enterobacteriaceae of the lactating doe was less than 50%, we excluded the maternal doe/kit couple from the analysis.The antibiotic susceptibility of TM in which 80 mg of frozen feces were processed according to the manufacturer’s instructions . The microbial DNA was also extracted from the three donor does at 16 days for ARG detection. The DNA concentration was measured using the Quant-iT TM PicoGreen TM dsDNA Assay Kit according to the manufacturer’s protocol  and subsequently diluted to 10 ng/μl.The microbial DNA was extracted at weaning from the three donors, the 37 dams and the 37 kits using the ZR-96 Fungal/Bacterial DNA KitTM Integrated Fluidic Circuit (IFC). Pre-amplification of the samples, chip loading and qPCR reactions in nanoliter volumes were performed according to the manufacturer’s protocol. A pre-amplification step was applied to all samples for all primer sets except the Zhu16S primer set. Briefly, 13 ng of total DNA were submitted to 14 PCR cycles using the PreAmp Master Mix (Fluidigm) and a mix of primers . Pre-amplified samples were 5-fold diluted after an exonuclease treatment. The diluted pre-amplified samples and the primer sets were loaded in a 96.96 IFC using an IFC Controller HX (Fluidigm). The Biomark thermal protocol was as follows: a thermal mix step , a hot start , 35 cycles of PCR , and a final melting phase (60°C to 95°C). The list of primers can be found in High throughput real-time qPCR was performed using the Biomark microfluidic system from Fluidigm  using a 96.96 Dynamic ArrayThe V3V4 region was amplified from the 77 purified genomic DNA with the primers F343 and R784  using 30The statistical analysis was performed with R 3.3.1 . The sigt-test. In other words, the decrease observed in the fecal pellets was compared to the decrease observed in the suspension for each ARG. The difference of the percentage of antibiotic-resistant Enterobacteriaceae between the different groups of lactating dams or groups of kits was also tested using the Kruskal-Wallis test, but the multiple testing was performed with the pgirmess package (version 1.6.9) in order to obtain a two-tailed p-value since we assume that the percentage of antibiotic-resistant genes decreases.The difference in the abundances of ARGs between the dams in the eight groups was tested using the Kruskal–Wallis test with the Benjamini–Hochberg correction of the two-tailed Dunn’s test for multiple testing since ARGs could increase or decrease. The efficiency of fecal pellets vs. the corresponding suspension in decreasing ARGs was tested using a paired The richness was estimated by the Chao1 estimate using the estimateR function (R package vegan 2.4-2), which was then analyzed by a one-way analysis of variance (ANOVA) to detect an impact of the group. The significance of the separation observed on the nMDS plot using the Bray-Curtis dissimilarity was tested using pairwise permutational multivariate analysis of variance with distance matrices on the square-root-transformed abundances of the operational taxonomic units (OTUs) with the adonis function, after confirming the homogeneity of the variance though the betadisper function (R package vegan 2.4-2). Two samples out of 37 were ignored because of aberrant profiles (kit10 and kit28).In order to quantify the immigration rates under the assumptions of the neutral model, we used the script provided by X- and an Y- vector of values by quantifying the non-randomness of points in a two-dimensional space. This index was calculated on all the OTUs with more than 10 counts on average, which represented 104 OTUs. A relationship was considered significant if its MIC value was observed in less than 1% of the 37950 MIC values across 100 randomized datasets. In our case, the MIC threshold values were 0.66365 for the kits and 0.65429 for the dams.The linear and the non-linear relationships between the OTUs and the ARGs were characterized by the maximal information coefficient (MIC) index . BrieflyAs expected, many antibiotic resistance genes are reduced in does from farms that have banned antibiotics for several years (referred to as “donor” does below). Based on the 37 samples from the lactating dams and the six samples from the donor does, half of the ARGs , whereas suspension I44 reduced the abundance of ermG by half (mefA (macrolide efflux pump), which explains why kits fed with suspension I41 had 312-fold more mefA than the control kits.When inoculating the kits with a microbial suspension every second day between day 2 and 29, suspension I43 reduced eight antibiotic resistance genes . In addition, the fecal pellets from Doe44 decreased the carriage of three different ARGs , whereas the corresponding suspension only changed ermG.Adding the fecal pellets to the nest is slightly more efficient than oral ingestion to drive the competitive exclusion. Indeed, six ARGs decreased in both kits exposed to suspension I43 and to the fecal pellets of Doe43, but the decrease was more drastic for kits exposed to pellets , while suspension I44 shows a trend that only becomes significant when performing a second trial  but the fecal communities of the kits in contact with fecal pellets from Doe43 and Doe44 differed from the communities observed in the control kits, unlike the fecal communities of kits in contact with fecal pellets from Doe41 . Finally, tet32 did not coexist with OTU20 belonging to the Lachnospiraceae family  associated with OTU95 belonging to the Clostridales order in which ermB determinants were previously detected in Clostridium .In does that were analyzed separately to avoid spurious relationships , the onlstridium  and in Rnococcus . It shouEnterobacteriaceae are tested for tetracycline and sulfonamide resistance using classic culturing techniques.In our study, we modified the microbial community that colonizes the gut of the kits using microbial fecal communities from foreign does by either providing them as anaerobic suspensions or as fecal pellets in the nest before weaning. The “donor” does are from farms that have banned antibiotics for at least 2 years. The feces are collected from the kits at weaning, at which point 26 antibiotic-resistant genes are evaluated via quantitative PCR, the gene coding for the small ribosomal subunit is sequenced, and the cultivable Enterobacteria in the feces of the kits mirrors the ratio observed in lactating dams. It should be noted that this ratio does not depend on age, unlike the abundance of the OTUs, as previously described in rabbits . Predicting the exclusion of targeted genes from complex communities is a critical milestone that requires additional information about the microbiota performing the exclusion. Indeed, it is challenging to predict the outcome of the exclusion based on the abundance of the ARGs in the donor doe and the lactating dam  or to small amounts of bacteria transmitted before birth, as observed in human . UnfortuSalmonella for decades, which probably also decreased the carriage of E. coli producing extended spectrum beta-lactamase as a side-effect .Enterobacteriaceae isolates for antibiotic resistance. VD, CSA, OZ, and AB-M drafted the manuscript. All authors edited and approved the manuscript.OZ, CSA, VD, and AB-M designed the study. CSA and SS extracted the microbial DNA, chose the primers, and analyzed the microbial sequences. LC uploaded the sequences. CSA and OZ carried out the statistical tests. VD and NA screened the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture. The skeletal muscle neuromuscular junction (NMJ) is a highly organized synapse formed between a motor neuron (MN) axon and a muscle fiber. It is designed to transmit efferent signals from projecting MNs to muscle fibers in order to actuate fiber contraction. Nicotinic acetylcholine receptors (AChRs) clustered at the NMJ’s postsynaptic muscle fiber membrane mediate this signal by binding acetylcholine (ACh) neurotransmitters released from vesicles at the presynaptic MN axon terminal. AChRs are ligand-gated ion channels composed of five protein subunits. During development the gamma subunit in embryonic AChRs is replaced by an epsilon subunit in the adult synapse . PreviouCongenital myasthenic syndrome is one of the most prevalent genetic diseases of the NMJ and commonly arises from mutations in one of the AChR encoding genes . The vasHere we report a method integrating architectural cues with co-culture techniques to create an environment conducive to the de novo formation of the adult human NMJ in as early as two weeks. In side-by-side studies of muscle fibers cultured in 2D, we show that the 3D culture system enables long-term maintenance of maturing muscle fibers in culture. It supports the formation and morphological maturation of AChR clusters primed for synaptogenesis and the de novo transition from the embryonic to the adult NMJ composition upon contact with MN endplates. We confirm formation of functional NMJ connections by imaging muscle fiber calcium transients and capturing electrophysiological recordings in response to glutamate-induced MN firing and demonstrate that treatment with inhibitors targeting pre- and post-synapse function block this firing. We show that the 3D co-culture platform, and not a 2D co-culture system, supports the transition from the embryonic to the adult AChR, thereby enabling the functional assessment of the adult neuromuscular junction in vitro. We present data aligning with prior studies showing that epsilon functional activity is regulated post-transcriptionally , and in We performed a side-by-side comparison of human skeletal muscle fiber populations derived in standard 2D culture versus 3D culture and uncovered differences in fiber maturation and AChR clustering . We estaImmunofluorescence analysis of the muscle contractile protein sarcomeric α-actinin (SAA) revealed the uniform alignment of striated muscle fibers along the tension axis in the 3D tissues , while 2In support of our molecular characterization, 3D human muscle tissues were capable of generating active force in as early as 10 days of differentiation as evidenced by spontaneous twitches , which wTo evaluate the calcium handling capacity of 3D muscle fiber cultures, we transduced human muscle progenitor cells with lentiviral particles encoding GCaMP6 , a sensiTo evaluate the electrophysiological characteristics of single muscle fibers in 3D cultures, muscle progenitor cells were stably transduced with a light-gated ion channel, channelrhodopsin-2 (ChR2), driven by an EF1α promoter . 3D musc2F). Further, our analyses revealed a greater number of AChR clusters per muscle fiber established in 3D compared to 2D culture  stimulation. Analysis of pre- and post-stimulation videos, to identify glutamate responsive fibers and substract spontaneously active fibers, indicated an average maximum functional connectivity length of 1042.7 ± 104.5 μm at this time-point. As expected, the number of innervated fibers decreased as the distance from the MN cluster increased .Finally, we performed electrophysiological recording to directly address the functional properties of the neuromuscular junctions. Using current clamp, we observed spontaneous endogenous endplate potentials (EPPs) from single muscle fibers that were proximal to the MN cluster , which wTogether, these studies indicate that 3D neuromuscular co-cultures support efficient functional innervation that occurs faster than previously reported for 2D neuromuscular co-cultures .Next, given the high degree of innervation achieved in our neuromuscular co-cultures, we hypothesized that the 3D model might be capable of supporting the gamma (embryonic) to epsilon (adult)-subunit switch that was not observed in 2D human neuromuscular co-cultures . SelectiWe next evaluated AChR epsilon expression at the protein level and found that it was upregulated in 3D co-cultures, but not in 2D co-cultures . The uprWe then sought to determine if the 3D human neuromuscular co-culture system was suitable for modeling congenital myasthenic syndromes caused by mutations in CHRNE by blocking the AChR-epsilon subunit using Waglerin-1 (WTX); a peptide that selectively binds and blocks the epsilon subunit of the muscle AChR . The AChCollectively, this data suggests that the 3D neuromuscular co-culture platform allows for rapid and easy modeling and study of diseases impacting the adult human NMJ.To demonstrate the tractability and robustness of the 3D neuromuscular co-culture system to study human disease, we treated co-culture tissues with IgG isolated from three patients afflicted with AChR-targeted myasthenia gravis  to modelThis study demonstrates the simplicity of implementing the 3D neuromuscular co-culture system to the application of modeling a human NMJ disorder in culture.Here we report a simple method to co-culture 3D human skeletal muscle fiber tissues together with human PSC-derived MNs, while performing comparative studies to uncover biological inquiries and processes that are enabled by the availability of a 3D human neuromuscular culture system. Our focus is distinct, but complementary to recent work reporting strategies to co-culture hPSC-derived skeletal muscle progenitors and motor neurons in 3D , and thaOur side-by-side comparison of human skeletal muscle fiber cultures in 2D and 3D indicates the structural and functional advantages of a 3D culture model over currently available 2D systems. The value of 3D culture is reported in previous studies for other organs , and in Consistently, electrophysiological recordings of single muscle fibers in these neuromuscular co-cultures detected putative endogenous and glutamate-stimulated EPPs, suggesting that MNs form functional neuromuscular junctions, similar to that observed in in vivo mammalian models. However, we will note that in the absence of pre- or post-synaptic blocking studies during these recordings, we can speculate, but not conclude definitively, that the activity we recorded was in fact evoked by glutamate stimulation. The properties of action potentials recorded in these cultures is consistent with the possibility that the 3D human neuromuscular co-cultures are not fully mature. This observation concurs with the relatively small muscle fibers, and indicates that additional chemical or physical cues are necessary to mature the tissues further. We saw a large action potential-like response in 1 out of the seven fibers we assessed. This suggests that although the NMJs are functional, exhibit endogenous activity, and that motor neurons respond to glutamate application by increasing the basal rate of neurotransmitter release, most motor neurons are still in an immature state and do not trigger synchronous neurotransmitter release in response to glutamate application. This could be at the level of action potential generation in response to glutamate application, or converting action potentials to synchronous release at the pre-synapse. We anticipate increasing culture time, providing electrical stimulation, and/or adding trophic or synaptogenesis factors might improve the maturity of the neuromuscular co-culture and their connections.Alternatively, our electrophysiological conclusions may simply reflect the technical challenges we faced in the course of our recordings . In general, analyses at the single fiber level require the user to develop or implement tools or adapt the protocol to improve feasibility. Electrophysiological recordings in the co-culture system are feasible, but highly challenging for a number of reasons. First, spontaneous or induced muscle tissue contractions in 3D neuromuscular co-cultures frequently resulted in the loss of pipette contact with the cell membrane during recordings. As a result, recording where glutamate is added to the culture bath as a stimulation method, which elicits tissue movement via multiple muscle fibers contracting in unison  only in the context of 3D neuromuscular co-cultures.2) and that 3D neuromuscular co-cultures exhibit spontaneous endogenous endplate potentials  were obtained from the multifidus muscle of patients undergoing lumbar spine surgery. Primary myoblast and fibroblast-like cell lines were established and maintained as previously described (+) and fibroblast-like cell (CD56—) populations were separated and purified using fluorescence-activated cell sorting (FACS) and maintained on collagen coated dishes in growth medium. Subsequent experiments utilized low passage cultures (P4—P9).Small skeletal muscle samples  and CD56— (5%). For Geltrex culture dish coating, 1 mg of Geltrex was resuspended in 12 mL of ice-cold DMEM and 1 mL was transferred to each well of a 12 well plate. Plates were incubated at 37°C overnight. DMEM was aspirated the next day just prior to cell culture. 3 × 106 cells resuspended in bFGF-free myoblast growth media . After curing at 50°C for at least 3 hr, another 750 μl of liquid PDMS was added to each well and a laser cut, dumbbell shaped piece of acrylic  was submerged in the liquid PDMS. Plates were then placed within a vacuum chamber for a minimum of 10 min to remove bubbles from the liquid PDMS. The PDMS was cured by incubating the plates in a 50°C oven for 3 hr. Acrylic pieces were then removed from the PDMS, leaving a dumbbell-shaped depression in the PDMS, and two pieces of Velcro fabric were affixed at each end of the channel using liquid PDMS as glue . Each weRemark: The integrity of nylon hooks, as anchor points, on the Velcro pieces is critical for the successful culture of 3D tissues and preventing their immature rupture. As such, we recommend careful inspection of the Velcro pieces before to ensure latch and hooks are without defects, and after to ensure pieces are well adhered in the dish. These are critical steps pre-tissue seeding to ensure successful tissue remodeling and culture.+ myoblasts (95%) and CD56— fibroblast-like cells (5%) were incorporated into tissues. Briefly, cells at these defined ratios were resuspended in the hydrogel mixture  human skeletal muscle tissues were generated in culture as previously described  with the mixture  in the a mixture  lacking  mixture  containi5 cells / cm2 in E8 medium containing 10 μM ROCK inhibitor  in 6-well polystyrene tissue culture plates coated with 100 μg / mL poly-L-ornithine  and 8 μg / well VTN-NC (gift from Dr. James Thomson). hESC were differentiated to OLIG2+ progenitors in E6 medium containing the same insulin concentration as in E8 medium as previously described , 100 nM purmorphamine (Tocris), and 100 ng/mL sonic hedgehog (R and D systems). To push neuronal maturation before myoblast co-culture, 5 μM DAPT (Tocris) was added from days 8–14.Motor neurons were specified from WA09 hESCs  as previously described . Brieflyescribed . For dif+ progenitor cells were specified following a previously described method , 5 ESC-derived motor neuron clusters were detached and transferred to the muscle cell culture plates using a 1 ml pipette tip in myoblast media lacking bFGF, but now containing 10 ng / ml brain derived neurotrophic factor (BDNF) and 10 ng / ml glial cell line derived neurotrophic factor (GDNF). Mid-sized clusters (150 to 300 μm in diameter) were visually identified and selected for transfer. 24 hr later the media was removed and replaced with myogenic differentiation media  supplemeFor 3D neuromuscular tissues, 3D skeletal muscle tissue cell/hydrogel suspension was prepared as described above. Motor neuron clusters were transferred manually to the cell/hydrogel suspension at a ratio of 5 clusters per tissue. Thrombin was added and tissues were seeded into dumbbell-shaped molds as described above. Myoblast growth media lacking bFGF, but containing 2 mg / mL 6-aminocaproic acid , 10 ng / mL BDNF, and GDNF was added to tissues. Two days later the culture media was exchanged to myogenic differentiation media  and supphttps://portals.broadinstitute.org/gpp/public/resources/protocols). The cell population was then sorted for GFP expression to enrich transduced cells and were then further expanded in myoblast growth media . Following fixation, samples were incubated in blocking solution  for at lMyofiber size was measured by assessing 40X magnification confocal images of 2D and 3D cultures immunostained for sarcomeric α-actinin. 2D muscle culture images and flattened z-stack images of 3D muscle tissues were analyzed to quantify the diameter of each muscle fiber using the NIH ImageJ.3D tissues were collected at the indicated time points and flash frozen in liquid nitrogen, while 2D cell cultures were directly lysed in RIPA buffer  and flas2 in area, were captured at a minimum of 6 random locations per sample. AChR cluster outlines in each 40X image were generated using the ImageJ particle analyzer. Clusters smaller than 5 μm2 were excluded from analysis. AChR cluster outline drawings were binarized to facilitate downstream analysis. To assess cluster number, AChR cluster were quantified for each image and was then normalized to the number of sarcomeric α-actinin+ fiber units present in the quantified image. To assess cluster area, the area of each individual AChR cluster was measured and averaged for each experiment using NIH ImageJ software. Fractal analysis was performed on α-bungarotoxin stained sample images to quantify AChR cluster morphological differences across the different culture conditions. After binarization of the AChR cluster outline drawings, lacunarity, a measure of gappiness and heterogeneity in a shape, was measured for each AChR cluster using the NIH ImageJ FracLac plug-in. The Sub Sample and Particle Analyzer method was used with FracLac along with default settings and four grid locations. Lacunarity was measured for each AChR cluster within an experiment and then averaged.α-bungarotoxin staining was performed to visualize and quantify the number, size, and morphology of AChR clusters in 2D cell and 3D tissue cultures. Briefly, fixed tissues were incubated with 5 nM of Alexa Fluor 647 conjugated α-bungarotoxin for 30 min to label AChRs. Samples were then washed with PBS and 40X images, all 0.1 mmTo ensure accurate and reproducible conditions for electrical stimulation, a custom-made stimulation chamber was produced using a 35 mm petri-dish, two carbon rods, and platinum wires. Before each use the stimulation chamber was sterilized using 70% ethanol. At day 14 of differentiation, an individual tissue was transferred to the chamber and covered in differentiation medium. Platinum wires were hooked up to a commercial function generator (Rigol DG1022U). A Rigol DS1102E digital oscilloscope was used to confirm the frequency and amplitude of signals before connecting the pulse generator to the platinum wires. 3D tissues were stimulated using square pulses with 20% duty cycle, 5V amplitude (field strength of 1.67 V/cm), and the reported frequencies.+ sorted human myogenic progenitor cells were transduced with a lentiviral vector encoding the fluorescent calcium indicator GCaMP6 driven by the muscle specific gene MHCK7 (AddGene plasmid #65042). Cells were then sorted to purify the infected cells based on GFP expression. Human skeletal muscle progenitor cultures expressing GCaMP6 were imaged using an Olympus IX83 microscope equipped with modules to control the temperature and CO2 concentration. Videos were recorded at 4X magnification at 12 frames per second under physiological condition (37°C and 5% CO2) in differentiation media using an Olympus DP80 dual CCD color and monochrome camera and CellSense software. Acetylcholine (BIO BASIC) was reconstituted to produce a 100 mM stock solution in PBS and was diluted to the final working concentration (as specified in the text) by addition directly into the culture chamber.CD56In glutamate stimulation studies, L-glutamate (Abcam) was first reconstituted to 100 mM in 1equal NaOH and then further diluted in HBSS (Gibco)/DMEM to produce a 100x stock solution (5 mM). For AChR epsilon subunit blocking studies, Waglerin-1 (Smartox Biotechnology) was prepared as a 100x stock solution in PBS and was added to cultures at a 1 μM working concentration 10 min prior to stimulation.To assess the effect of Waglerin-1 on glutamate-stimulated calcium transients, a video was recorded for each tissue before and after glutamate stimulation. Video segments, equal in length, representing pre- and post-glutamate GcAMP6 signals were each projected into a 2D image. The 2D projected images were then subtracted to eliminate spontaneously active fibers from our analysis. Background from different imaging sessions were normalized. In To assess the effect of BOTOX  and d-tubocurarine (Sigma) treatments on glutamate-stimulated calcium transients, co-cultures were treated with BOTOX (1 U/ml)  and d-tuC and 5% CO2). To quantify neuromuscular tissue contraction, videos were assembled into stacks using ImageJ software and 3 regions of interest were traced within each stack. Maximum movement distance for each trace was determined and averaged for each sample. Data are presented as movement (distance) in pixels.To assess the contraction of neuromuscular tissues following glutamate and acetylcholine stimulations, co-cultures were stimulated under the indicated experimental conditions and videos were recorded at 4X magnification at 12 frames per second under physiological conditions (37°+) on the subtracted image. The number of active fibers in each circle were then quantified to determine the number of fibers in each concentric circle ‘bin’. Binned data from three independent experiments were then reported on a histogram to report the average number of glutamate responsive fibers as it relates to the distance (i.e. concentric circle bin) from the motor neuron cluster.In this studies, neuromuscular co-cultures were generated using GCaMP6 transduced human myogenic progenitor cells and a single motor neuron cluster. At week two of co-culture, tissues were stimulated by a 50 μM L-glutamate solution diluted in DMEM. Videos were captured at a frequency of 12 frames per second for at least 15 s before and after stimulation. Videos were processed exactly as described above  to eliminate the spontaneously active fibers from the analysis. The location of the motor neuron cluster was identified from a bright field image, which was used to outline the structure with a circle in the epifluorescent images. Using NIH ImageJ software, concentric circles, each 100 μm larger in radius than the prior, were outlined around the motor neuron cluster until the circles encompassed all the glutamate responsive fibers , filled with 3M KCl. Membrane potential was recorded in the current clamp configuration using a Digidata 1440A and MultiClamp 700 A amplifier . Data were digitized at 10 kHz and filtered at 2.6 kHz. Data were quantified using MiniAnalysis (Synaptosoft). Each fiber was allowed to recover for a few minutes, to allow its resting membrane potential to stabilize before recordings were performed. For electrophysiological recordings following optogenetic stimulation, 3D muscle tissues were generated using human skeletal muscle progenitors transduced with a lentiviral vector encoding humanized ChR2 with H134R mutation fused to EYFP and driven by EF1a (AddGene plasmid #20942). Cells were sorted to purify the infected cells based on the EYFP signal. Optogenetic stimulation was performed using blue LED  with a wavelength of 470 nm, and controlled by the Axon amplifier software. In glutamate stimulation experiments, glutamate was pipetted by hand into the edge of the bath and allowed to diffuse to the tissue. For all recordings, the bath solution was standard DMEM (Gibco).Remark: To facilitate electrophysiological recordings, 3D neuromuscular tissues were removed from their culture well by popping the entire circular piece of PDMS containing the channel and co-culture tissue out of the 12-well plate and transferring it into a 35 mm culture dish. Addition of the neurotransmitter glutamate generates ripples and disturbs the equilibrium in the culture bath that may lead to the loss of sharp microelectrode recording. Therefore, the bolus of the neurotransmitter should be added from the side of the culture bath gently to then allow for its diffusion the tissue.Remark: Spontaneous contractions of the neuromuscular tissues complicate the sharp microelectrode recordings. Neuromuscular tissues are most spontaneously active once removed from the incubator. As such, resting the tissue at room temperature for 5–10 min after transferring from the 37 C incubator reduces the spontaneous activity and facilitates the sharp microelectrode recordings.Serum from three patients diagnosed with Anti-AChR MG  was coll+ tissue as described above. Our analysis indicated that the area of glutamate responsive tissue increased from ~2% at Day 11 to more than 13% on Day 14, arguing against glutamate-induced cytotoxic effects arising from our experimental methods.In a subset of experiments, we performed serial glutamate stimulation experiments in which we stimulated the same neuromuscular co-culture on Day 11 and then Day 14 of culture with a 50 μM L-glutamate solution and measured the area of GCaMP62+and + Ca2+) for 20 min to enable dye loading. Clusters were washed with HBSS for at least one hour at room temperature before imaging. Samples were imaged using an IX83 Olympus confocal microscope with FV-10 software at physiological conditions (37°C and 5% CO2). MN clusters were then stimulated with either high potassium solution (60 mM in PBS), L-glutamate (50 μM in HBSS), or control solutions (HBSS or PBS) while acquiring time-lapse video sequences. Videos were analysed for fluorescence intensity before and after stimulation at each indicated time-point using NIH ImageJ software.Motor neuron clusters were separated from undifferentiated single cells using Accutase (ThermoFisher) and transferred to a Geltrex coated 6-well plate on Day 14 of differentiation. Clusters were then cultured for an additional week in E6 media supplemented with 10 ng/mL BDNF and 10 ng/mL GDNF, to permit the regrowth of the neurites, and were then labeled with the FM 1–43 styryl dye (Molecular Probes) following the manufacturer’s instructions. Briefly, MN clusters were stimulated using high potassium solution (60 mM) and incubated with FM 1–43 (2 μM) in HBSS . cDNA was reverse transcribed from 400 ng of RNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). For quantitative real-time PCR (qRT-PCR), CHRNE and CHRNG primers were acquired from Bio-Rad and reactions were run according to manufacturer’s protocol on the Roche LightCycler 480 (Roche) using LightCycler 480 SYBR Green I Master (Roche). All results were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gene expression is reported in % of GAPDH expression ±SEM. To assess agrin gene expression in differentiated MNs, cDNA samples were prepared from three consecutive MN differentiations. Genes were amplified using Arktik thermal cycler according to the manufacturer’s protocol (ThermoFisher). PCR amplification products were analyzed on a 2% agarose gel with SYBR safe DNA gel stain (Invitrogen). GAPDH gene expression served as the loading control. All oligo sequences are summarized in Each study in this manuscript was performed using three primary myoblast lines derived from three separate muscle patient donors . Each experiment within a study was set-up with cells from a separate muscle donor and included at least n = 3 technical replicates. Exceptions include #, $) indicates no significant differences.Statistical analysis was performed on data obtained from technical replicates using GraphPad Prism 6.0 software. Statistical differences between experimental groups were determined in most studies by unpaired t-test. Exceptions to this are as follows: Two-way ANOVA followed by Tukey’s and Sidak’s multiple comparisons were performed in  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Andrew Brack as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Marianne Bronner as the Senior Editor.Thank you for submitting your article "A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The manuscript from the Gilbert lab provides the first demonstration of a robust method to generate mature muscle fibers with functioning 'adult' NMJ in vitro. This has been a significant hurdle for many years. This new approach will have significant utility for basic scientists working in the muscle or NMJ field, and those that model disease in vitro. The experiments have been performed to the highest standard, the data are compelling, the methods thorough, and the statistics appropriate. This work is a significant technical advance.Reviewers felt that the authors provide compelling evidence for the use of 3D muscle cultures. While there is general excitement for the manuscript, there were two main issues raised. 1) How easy is it to adopt this technique? It was mentioned that readers would benefit from a more transparent discussion on the method, outlining the difficult steps and the pitfalls that drive its failure/success. 2) Further evidence to describe the maturation status of the NMJs. Is activity required for the maturation of the muscle fibers?Below are the main points to consider. These points are given more context within the reviewer's individual comments.1) How easy is this method to be adopted? Please provide information outlining the ease of adaptability and the technical hurdles that are critical for success.2) More data to determine the maturation of the NMJs:- The authors should stain individual NMJs in their 3D system with antibodies against the other proteins  that they study in bulk cultures. Do they get more fully formed NMJs?- Is there any difference in the NMJs between two systems when investigated more at the level of single NMJs ?- Does curare block all of the activity?- The increase in EPP frequency is not obvious. The authors should apply TTX to the cultures to at least distinguish between EPPs and mEPPS. Do the authors see action potentials prior to glutamate application?3) Are there lessons that can be learnt from the 3D cultures that could be implemented to improve 2D cultures? If the authors have data that could be incorporated within the 2-month revision window, please do so. If not, the manuscript would benefit from a discussion on this issue.Reviewer #1:The manuscript from the Gilbert lab provides the first demonstration of a robust method to generate mature muscle fibers with functioning 'adult' NMJ in vitro. This has been a significant hurdle for many years. This new approach will have significant utility for basic scientists working in the muscle or NMJ field, and those that model disease in vitro. The experiments have been performed to the highest standard, the data are compelling, the methods thorough, and the statistics appropriate. This work is a significant technical advance.I have one request for the authors, which stems from the fact that the take away message is the ease of the method. And while the data support this notion, I would ask the reviewers to provide more information (in commentary or data) as to where the limitations or difficulties in the method lie. For example, what is the day-to-day variability between neuromuscular samples, or the variability between dishes? Is there uniformity across time/samples/experimentalist?In essence, it would be helpful to any adopter of this method if they understood the limitations and potential roadblocks.Reviewer #2:eLife and should be published after suitable revisions.The paper by Bakooshli et al. presents a new system to study human muscle development, neuromuscular junction (NMJ) formation, and, perhaps, neuromuscular disorders. The paper is fairly comprehensive and mostly quite well-done. I think it is suitable for I have three general comments and then several specific questions detailed below.First, the authors have compelling data illustrating differences in the speed of muscle development. Can any lessons be learned  from 3D that could be applied to 2D cultures?Second, while the authors do an admirable job of comparing their new 3D systems with a more conventional 2D systems , they should try to objectively present the advantages AND disadvantages of 3D. First of all, it's not clear to me how easy the system described in this manuscript would be to establish in a non-engineering based lab. A guide to its use or an outline of the type of future developments that would make this system more routine would be useful. Many of the authors' measurements are made on "bulk" cultures, with fewer being done on individual fibers or individual NMJ's. So, certainly one downside may be that 2D does make some of that more straightforward My final general comment is that the myasthenia gravis work seems like an almost unnecessary add-on. Was this done to compare the current system to that used by Steinbeck et al., 2016?Reviewer #3:In vitro studies of skeletal muscle can be problematic because the in vivo function of these cells requires interaction with multiple other cell types including motor neurons, however these interactions are difficult to replicate ex vivo. This article describes a method for generating de novo differentiated muscle fibers 3D culture in vitro, coupled with generation of pluripotent stem cell-derived motor neurons, which will self-assemble into functional units in coculture. To validate the technique, the authors showed that these muscle fibers express myosin heavy chain and acetylcholine receptor isoforms specific to mature adult muscle  and demonstrated that the neuromuscular synapses (NMJs) respond in a physiologically appropriate fashion to electrical and chemical stimulation, as measured by muscle contractile activity, calcium flux, and electrophysiology. These muscle fibers and motor neurons were generated from human cells, so as a final proof-of-principle the authors treated the matured neuromuscular units with serum from patients with myasthenia gravis (MG), an autoimmune disease which targets the mature isoform of NMJ receptors and diminishes their function. Within two weeks of treatment, they observed alterations in the in vitro generated NMJs mimicking clinical signs in patients, supporting the potential utility of these in vitro cultures for modeling human disease.This paper is a fairly straightforward paper; the experiments are well designed and documented and the paper itself is clearly written. The authors are correct in stating that there is an unmet need in the field for an in vitro model of the mature NMJ, and the culture system they describe is better than anything currently in use in the field. As this is primarily a technique paper, I don't think additional studies beyond what is already included are necessary, although there are several that could be done which come to mind- if this is published, I think it will be of significant use to the field and those experiments will be carried out by this group and others. Reviewers felt that the authors provide compelling evidence for the use of 3D muscle cultures. While there is general excitement for the manuscript, there were two main issues raised. 1) How easy is it to adopt this technique? It was mentioned that readers would benefit from a more transparent discussion on the method, outlining the difficult steps and the pitfalls that drive its failure/success. 2) Further evidence to describe the maturation status of the NMJs. Is activity required for the maturation of the muscle fibers?Below are the main points to consider. These points are given more context within the reviewer's individual comments.1) How easy is this method to be adopted? Please provide information outlining the ease of adaptability and the technical hurdles that are critical for success.We thank the reviewers for the opportunity to provide more detail related to our personal experiences with this co-culture method and platform. Indeed, this information is key to ensuring widespread adoption, together with our proactive efforts in hosting labs from across the world for hands-on training. We now provide additional detail in the Materials and methods and we expand on the point of technical hurdles and system adaptation pointers in the Discussion.See also detailed response to reviewer #1.2) More data to determine the maturation of the NMJs:- The authors should stain individual NMJs in their 3D system with antibodies against the other proteins  that they study in bulk cultures. Do they get more fully formed NMJs?- Is there any difference in the NMJs between two systems when investigated more at the level of single NMJs ?To address this issue, we set up cultures and performed immunostaining on 2D and 3D muscle alone cultures as well as 2D and 3D neuromuscular co-cultures. We did not observe any examples of AChR clusters co-localizing with either rapsyn or MuSK proteins in our 2D muscle-alone or neuromuscular cultures .We observed a single incidence of MuSK protein co-localization with an AChR cluster in our 3D muscle-alone cultures  and no incidences of rapsyn co-localization with AChR clusters in our 3D muscle-alone cultures.By comparison, the prevalence of rapsyn and MuSK co-localizing with bungarotoxin stained AChR clusters was substantially higher in 3D neuromuscular co-cultures. Two representative examples where we observed co-localization of rapsyn  and MuSK proteins  with MN neurites at bungarotoxin stained AChR cluster sites are shown. However, we note that not all the AChR clusters co-localized with MuSK and rapsyn in 3D neuromuscular co-cultures at the experimental time point we evaluated (Day 12).- Does curare block all of the activity?We use 25 μM treatment of d-tubocurarine in our system. At this concentration we observe over 99% blocking of changes in muscle fiber calcium transients post glutamate stimulation of motor neurons. This is matched with the results from Ko et al., 2019 were they observe a full block at 50 μM. In addition, we do not observe a change in calcium transients of d-tubocurarine treated (25 μM) myofibers stimulated with 100 μM, ACh (data not shown) which demonstrates the potency of d-tubocurarine blocking at this concentration. These data are consistent with previous reports  using similar concentration of curare and recent work from our laboratory deposited on BioRxiv (https://doi.org/10.1101/562819). We now make this point and refer to these citations in the text.- The increase in EPP frequency is not obvious. The authors should apply TTX to the cultures to at least distinguish between EPPs and mEPPS. Do the authors see action potentials prior to glutamate application?As (correctly) alluded to by all reviewers, we faced challenges in performing electrophysiological recordings. We did indeed set up neuromuscular co-cultures to address this concern, but in the end, ran into technical hurdles that precluded the gathering of data. As the protocol requires 5 weeks from beginning to end, we faced some pressure in collecting the data in the 2-month window, but we did try. As suggested by all reviewers, we now make clear in the Discussion challenges associated with performing e-phys in culture and offer suggestions to adapt methods for future users who wish to perform e-phys in this system.We have updated the text to faithfully reflect the data we have in hand. Specifically, we never saw spontaneous activity in 3D muscle-alone recordings , whereas, it was commonplace to capture spontaneous activity in 3D neuromuscular co-cultures . We now speculate (rather than conclude), that these spontaneous endogenous end plate potentials (EPPs) are caused by spontaneous release of ACh from the motor neuron axon terminals at the NMJs, and in the Discussion we acknowledge that without performing blocking studies, we cannot make this conclusion definitively. While our analysis indicated a glutamate evoked change in EPP frequency, we understand that the trace provided was less than satisfactory. We now provide more representative traces we collected in the course of studies aimed at capturing evoked potentials using e-phys . In the Discussion we acknowledge that without performing blocking studies, we cannot make this conclusion definitively.Action potential-like events were only observed post glutamate stimulation. We now make this point in the Results section.3) Are there lessons that can be learnt from the 3D cultures that could be implemented to improve 2D cultures? If the authors have data that could be incorporated within the 2-month revision window, please do so. If not, the manuscript would benefit from a discussion on this issue.We now address this point in our Discussion. See corresponding text below.Based on our comparative analysis, a major advantage of a 3D culture system is the ability to support the contractility of maturing myofibers, which then are the ideal template for motor neuron innervation in as early as 12 days of differentiation. In addition, myofibers form packed and aligned structures in a 3D system which is not observed in standard tissue culture plastic 2D cultures.As such, we expect that focusing efforts on modifying 2D cultures to control the microenvironment in ways that can accommodate myofiber contractility and myofiber alignment, might result in the formation of functional NMJs in vitro at earlier time-points, and perhaps even support the de novo embryonic to adult switch in AChR subunit type. Indeed, a recent report by Ko and colleagues  working with rodent myoblast and neural cells highlights a role for matrix topography and myofiber alignment as it relates to innervation efficiency in 2D culture, though more comparative analysis is required to understand how this type of approach impacts human NMJ formation in culture as compared to a 3D co-culture system.Reviewer #1:[…] I have one request for the authors, which stems from the fact that the take away message is the ease of the method. And while the data support this notion, I would ask the reviewers to provide more information (in commentary or data) as to where the limitations or difficulties in the method lie. For example, what is the day-to-day variability between neuromuscular samples, or the variability between dishes? Is there uniformity across time/samples/experimentalist?In essence, it would be helpful to any adopter of this method if they understood the limitations and potential roadblocks.We thank the reviewer for highlighting this point. Here we provide additional detail to address the issue of technical challenges and reproducibility. We have added text to the Materials and methods to provide tips for success. We also expand on technical limitations and suggest adaptations that will expand the usability of the system.1) While the muscle culture protocol consistently yields muscle tissues, the use of Velcro as an anchor points is not ideal. Glue-ing the Velcro into each side of the culture depression is time consuming and requires some practice. In some instances, for example if the Velcro hooks were broken and/or weak, a 3D muscle tissue might rupture at an early time point. As a result, the user must assess the quality of the anchor points thoroughly to ensure the successful formation and maintenance of the muscle tissues. To overcome this technical issue, we designed and produced a 96 well plate that enables reproducible bulk production of muscle microtissues in vitro that uses deflective rubber posts cast directly into the plate as ‘tendon’ anchor points, rather than Velcro .+ progenitor cells also impacts motor neuron cluster formation and differentiation yields. As such, potential adaptors need to test each new batch of differentiation factors and also ensure consistent seeding criteria of progenitor cells to ensure high differentiation yields. Finally, it should be noted that excessive shearing of Olig2+ cells upon thawing and plating, reduces the differentiation efficiency in subsequent steps.2) Challenges associated with specifying pluripotent stem cells to the motor neuron lineage can delay planned experiments. This is a particular hurdle given that current motor neuron specification protocols are 2-3 weeks long. Common issues include the batch to batch variability and/or limited shelf-life of the motor neuron differentiation factors  which can have a significant impact on the success of the motor neuron differentiation protocol. The initial seeding density of Olig23) The co-culture protocol relies on collecting individual motor neuron clusters to mix together with muscle progenitor cells, as explained in the Materials and the methods. This is advantageous as it enables the user to collect a defined number of uniformly sized clusters that are free of any contaminating, undifferentiated cells. However, this step is very labor intensive and limits the throughput of the co-culture tissue production. Therefore, future work aimed at defining protocols for enzymatic collection of clusters followed by a stepwise straining process to support high throughput collection of uniformly sized motor neuron clusters is needed.4) To ensure easy fabrication of neuromuscular co-culture tissues, motor neuron clusters are mixed directly into the hydrogel mix containing the mononucleated muscle progenitor cells. While this allows for easy fabrication of neuromuscular tissues, the process precludes control over the final localization of motor neuron clusters as the neuromuscular tissues form. As such, the proximity of motor neuron clusters to one another, and the degree of innervation within co-culture tissues can vary a bit from one tissue to another.5) Compared to 3D muscle only tissues, we observed a lot of contractile activity in neuromuscular co-cultures, which we expect is due to the spontaneous firing of motor neuron cells present in co-cultures. Based on the literature, we surmise that the higher motor neuron induced muscle fiber activity contributes to the advanced contractile maturation of muscle fibers in our co-cultures , but in turn also led to higher rates of muscle rupture in co-cultures. Based on a recent report  highlighting the influence of plasmin, cathepsin L, MMP-2, and MMP-9 proteases on in vitro muscle tissue longevity, we propose that using an array of protease inhibitors, in addition to ACA, might aid in preventing contraction-induced neuromuscular co-culture demise.6) In general, analyses at the single fiber level require the user to develop tools or adapt the protocol to improve feasibility. For example, electrophysiological recordings in the co-culture system are feasible, but highly challenging for a number of reasons. First, spontaneous or induced muscle tissue contractions in 3D neuromuscular co-cultures frequently resulted in the loss of pipette contact with the cell membrane during recordings. As a result, recording where glutamate is added to the culture bath as a stimulation method, which elicits tissue movement via multiple muscle fibers contracting in unison (as seen in Video 7) were exceedingly challenging and as such, only successful in few events, as reported in the manuscript. However, this challenge can be overcome by performing targeted stimulation of single motor neurons with electrical or neurotransmitter stimulation, or by stimulating with blue light in the case of motor neurons genetically modified to express a light sensitive channel (e.g.) channelrhodopsin. In addition, identifying innervated muscle fibers is challenging. Using fluorescently labelled muscle cells  and motor neuron  cells will dramatically improve the success rate in identifying innervated muscle fibers in 3D neuromuscular co-cultures. Notably, the 3D nature of the co-culture system reduces the incidence of pipette breakage common in studies of plastic cultured myotubes. However, without added myotube maturation through contraction regimes or otherwise, it should be noted that myotubes in culture are somewhat smaller than those in adult animals, which can introduce some difficulty in recording. Other types of single fiber or single molecule analyses, such as immunostaining or time-lapse microscopy to follow a single acetylcholine receptor subunit, are more challenging in 3D as compared to 2D culture, but are feasible.Reviewer #2:The paper by Bakooshli et al. presents a new system to study human muscle development, neuromuscular junction (NMJ) formation, and, perhaps, neuromuscular disorders. The paper is fairly comprehensive and mostly quite well-done. I think it is suitable for eLife and should be published after suitable revisions.We thank the reviewer for acknowledging the value and quality of our work.I have three general comments and then several specific questions detailed below.First, the authors have compelling data illustrating differences in the speed of muscle development. Can any lessons be learned  from 3D that could be applied to 2D cultures?We now address this point in our Discussion. See corresponding text below.Based on our comparative analysis, a major advantage of a 3D culture system is the ability to support the contractility of maturing myofibers, which then are the ideal template for motor neuron innervation in as early as 12 days of differentiation. In addition, myofibers form packed and aligned structures in a 3D system which is not observed in standard tissue culture plastic 2D cultures.As such, we expect that focusing efforts on modifying 2D cultures to control the microenvironment in ways that can accommodate myofiber contractility and myofiber alignment, might result in the formation of functional NMJs in vitro at earlier time-points, and perhaps even support the de novo embryonic to adult switch in AChR subunit type. Indeed, a recent report by Ko and colleagues  working with rodent myoblast and neural cells highlights a role for matrix topography and myofiber alignment as it relates to innervation efficiency in 2D culture, though more comparative analysis is required to understand how this type of approach impacts human NMJ formation in culture as compared to a 3D co-culture system.Second, while the authors do an admirable job of comparing their new 3D systems with a more conventional 2D systems , they should try to objectively present the advantages AND disadvantages of 3D. First of all, it's not clear to me how easy the system described in this manuscript would be to establish in a non-engineering based lab. A guide to its use or an outline of the type of future developments that would make this system more routine would be useful.The 3D system culture platform requires a laser engraver (cutter) to prepare the dog-bone shaped molds to fabricate rubber channels in a standard 12-well plate. Otherwise, no specialized equipment or know-how is required. A detailed experimental procedure about the fabrication of the platform is provided in the Materials and methods section under the “PDMS mold fabrication for 3D human muscle tissue culture”.Many of the authors' measurements are made on "bulk" cultures, with fewer being done on individual fibers or individual NMJ's. So, certainly one downside may be that 2D does make some of that more straightforward While we highlight the advantages of the 3D culture compared to the conventional 2D system in our manuscript, the reviewer is undoubtedly right that certain aspects of muscle studies are more straightforward when done in a 2D culture. For example, image based analyses are often easier to perform in 2D systems.Another challenge with our 3D co-culture studies, as we elaborate on above, was to locate the individual fibers for electrophysiological recordings. Using fluorescently labelled cells would help to address this issue and would facilitate individual fiber analysis in a 3D system. Further, stimulating motor neurons locally using electrical, chemical, or optogenetic methods, as opposed to adding glutamate directly to the bath (as done in our studies), will mitigate issues of tissue movement that often accompanied lost recording opportunities.Despite these challenges, it is important to note that the development of NMJ formation is a time taking process which, by definition, includes formation of contractile myofibers and AChR clustering. Therefore, a 2D or 3D system capable of myofiber maintenance and contractile maturation is needed to study the formation of functional adult NMJs.My final general comment is that the myasthenia gravis work seems like an almost unnecessary add-on. Was this done to compare the current system to that used by Steinbeck et al., 2016?We acknowledge and understand this comment about our myasthenia gravis work. As one might expect, these experiments were included to appease a prior reviewer. We agree that it does not substantially add to the primary message of the manuscript, but as it does demonstrate the ease of modeling an autoimmune NMJ disease in the in vitro culture system in as early as 14 days of differentiation, we prefer that the work remain in the manuscript.


The overall success of a periapical surgery is assessed in terms of regeneration of functional periradicular tissues. The regenerative potential of platelets has been well documented. This article describes the use of concentrated growth factors (CGF), a new family of autologous platelet concentrates, as a sole material for bone regeneration after periapical surgery. 32- and 35-year-old female patients diagnosed with Ellis Class IV, an open apex in 11 with apical periodontitis in 11 and 12 and previously root canal-treated 31 and 41 with a chronic apical abscess, respectively, were managed with endodontic surgery. Subsequent to apicectomy and retrograde filling, the CGF fibrin block and membrane were used before suturing. There was uneventful healing during the immediate post-op and the subsequent follow-up periods. CGF is produced by a differential centrifugation process that results in the formation of a denser fibrin matrix richer in growth factors than those observed in PRF. Reasonable osseous healing was seen as early as 6-month follow-up, thereby recommending the use of CGF as an alternative to bone grafts and membranes in extensive periapical lesions to enhance bone regeneration and to decrease the healing time. Periapical surgery is the treatment of choice for teeth with persistent apical periodontitis when the conventional nonsurgical method fails to achieve the principal objectives . It has Platelet-rich plasma (PRP) and plasma rich in growth factors (PRGF) classified as the first generation of autologous platelet concentrates (APCs) have demonstrated promising results in the management of extensive periapical lesions, apicomarginal defects, and combined endo-perio lesions and also improved the quality of life of patients undergoing endodontic surgery . HoweverPlatelet-rich fibrin (PRF), considered as the second generation platelet concentrate and classified into L-PRF, A-PRF, i-PRF, CGF, PRFM, and Vivostat PRF . A systehttp://www.care-statement.org/) [Concentrated growth factor (CGF) is a newer second generation platelet concentrate that is prepared by centrifuging blood samples at alternating and controlled speeds using a special centrifuge. Differential centrifugation results in the formation of a denser fibrin matrix richer in growth factors than those observed in the PRF and PRP. To our knowledge, this is the first case report for the use of CGF alone in endodontic surgery. For describing the case reports, we have followed the checklist given in CARE guidelines (nt.org/) .A 32-year-old female patient was referred to the Department of Conservative Dentistry and Endodontics with chief complaint of discolouration of the upper right front tooth (tooth 11) for the past 6 months. Past dental history revealed a traumatic fall 5 years ago which involved the upper front teeth for which no dental treatment was sought for. Recently, as the discolouration gradually worsened, the patient sought dental treatment to restore esthetics.Intraoral clinical examination revealed a slightly discoloured 11 and chipping of the incisal edge with no presenting symptoms . Both 11μm, and 14 s) to assess the exact location, size, and extent and proximity of the lesion to anatomical structures.On radiographic examination , extensive periapical radiolucency involving the roots of 11 and 12 and an open apex of 11 was revealed . CBCT foThe preoperative measurements of the lesion as seen in CBCT in different planes can be seen in Figures A tentative diagnosis of an Ellis Class IV fracture and open apex in 11 with apical periodontitis in 11 and 12 was made. The differential diagnosis could be a chronic periapical abscess, periapical cyst, and periapical granuloma. The treatment plan was root canal treatment for 11 and 12. The patient was informed about the risks and benefits of the procedure and a written consent was taken.Under local anaesthesia  and rubber dam isolation, an access cavity was prepared in 11 and 12 with an Endo access bur . The working length was determined with an apex locator  and confirmed with radiograph. Cleaning and shaping was initiated with 45 K file , and apical preparation was performed till size 80 K file in 11. In 12, apical preparation was done till size 45 K file to full working length, after which step-back preparation was done till 80 K file. Routine root canal-shaping procedure was done along with copious irrigation using 3% sodium hypochlorite  and final flush with 0.9% physiologic saline . Calcium hydroxide medicament  was placed thrice for a period of 1 week each. As the root canals exhibited persistent discharge of exudates and due to two prognostic factors  which were unfavourable for this case [The root canals were obturated a day before the surgery using custom-made roll cone technique for 11 and conventional cold lateral compaction technique for 12.During surgery, after achieving adequate anaesthesia, a crevicular incision was placed from 22 to 14 with a vertical releasing incision on the mesial aspect of 14 to reflect a full thickness mucoperiosteal triangular flap. Cortical softening of the periapical bone was noted from regions 11 to 13. A bony window was created and thorough curettage was done .Apicoectomy was performed extending 3 mm into the canal space using no.702 tapered fissure burs (SS White burs) in 12. Apicoectomy was not done in 11 because of the presence of an open apex. Root end cavity preparation was done using zirconium nitride ultrasonic retro-tips  in 11 and 12. Subsequently, retrograde filling was done with Mineral Trioxide Aggregate (MTA Angelus® Brazil) . Later, A standard, disposable, two 10 mL nonanticoagulant glass tubes and a matching centrifuge device  were used. 20 mL of intravenous blood sample from the patient was placed in centrifuge tubes without anticoagulants and accelerated for 30 s, centrifuged at 2700 rpm for 2 min, 2400 rpm for 4 min, 2700 rpm for 4 min, and 3000 rpm for 3 min, and decelerated for 36 s to stop. All of these processes are adjusted automatically by “preprogramming” in the machine.From the three layers formed, the uppermost platelet-deprived fraction was removed with a sterile syringe. The layer in the form of a fibrin gel containing the CGF was separated from the red blood cell layer. The prepared CGF fibrin gel was placed inside the surgical site and covered with CGF membrane. The layer in the form of a membrane containing the concentrated growth membrane was held with a hemostatic clamp and separated from the RBC layer by using microsurgical scissors. The CGF layer is then placed in a condensing disc and compressed to convert to CGF membrane. Figures .Subsequent to CGF placement, the flap was closed with 3-0 vicryl sutures . Postoperative instructions were given and systemic antibiotics, analgesics, and supplemental 0.2% chlorhexidine mouthwash were prescribed.The postoperative CBCT at 1-year follow-up showed satisfactory healing with evident reduction in lesion size as shown in Figures A 35-year-old female patient reported mild swelling and pus discharge in the lower front region of the mouth for the past 2 months. Past dental history revealed trauma to lower anterior teeth 4 years ago, following which she underwent endodontic treatment. History suggested that there were multiple retreatments in the past for the current chief complaint.On clinical examination, 31 was discoloured with mild swelling on the labial aspect . RadiogrUnder local anaesthesia, the full thickness trapezoidal mucoperiosteal flap was reflected with vertical releasing incisions taken from the mesial aspect of 43 and 33. The surgical site was flushed with sterile saline after thorough curettage of the lesion . Followi3 and 0.08 cm3, respectively. The lesion size reduction was found to be 89.2%. In case 2, the preoperative and postoperative volume calculations were 0.1358 cm3 and 0.0101 cm3, respectively. The lesion size reduction was found to be 92.5%.At 1-year follow-up with CBCT, livewire segmentation using OSIRIX Version 9.5  was done to delineate the lesion from the healthy bone. In case 1, the preoperative and postoperative volume calculations were 0.7862 cmOur case reports are unique in that CGF was produced using the recommended centrifuge, and it was used as a sole material to understand its exclusive role in repair and regeneration after periapical surgery.During the preparation of APCs, the quality of the resultant product is greatly influenced by centrifugal characteristics like rotational speed (rpm), time (min), and centrifugation protocols. The centrifugation method for CGF is “preprogrammed” and the resultant CGF is stronger, thicker, and abundant with growth factors. The centrifuge is equipped with self-ventilation that prevents the temperature rise which helps to maintain the viability of enmeshed cells in the fibrin matrix. The dense three-dimensional network of the fibrin ensures slow release of growth factors. Park et al. affirmed that CGF consisted of thicker fibrinogen fibres per area unit and regular fibrinogen structures compared to PRF .β1 was released for one week whereas PDGF-BB for 3 weeks and identified that VEGF was one and a half times more in CGF than PRF [β1 had a slow release for 13 days [The presence of growth factors and leucocytes including also the few CD34+ circulating cells that are concentrated in a small volume is the probable reason for this success, since all the elements have been demonstrated to play an important role in vascular maintenance, angiogenesis, and neovascularization. Park et al. reported that TGF-than PRF . However 13 days . In a co 13 days .Growth factors act on target tissues and regulate a variety of cellular events including cell migration, proliferation, and differentiation. Borsani et al. have ascertained that CGF addition enhanced cell proliferation of fibroblasts, endothelial cells, and osteoblasts which are involved in angiogenesis, tissue remodelling and regeneration .There are two case reports in the literature which describe the utilization of CGF in periapical surgery. In the first, CGF was not prepared with the special centrifuge . In the Considering the encouraging result of these case reports, concentrated growth factors could be recommended as an alternative to bone grafts and membranes in extensive periapical lesions to enhance bone regeneration and to decrease the healing time. However, this case series is an initiative for application of CGF in endodontic surgery. The utilization of CGF in endodontics can be extended to revascularization procedures and endo-perio lesions also. However, the major limitation is that according to the CEBM level of evidence, the case reports have the lowest level of evidence. A well-designed randomized clinical trial is recommended to comprehend the long-term risks and benefits of using CGF in regenerative endodontics.


Autoclave manufacturing of fibre metal laminates, such as GLARE, is an expensive process. Therefore, there is an increasing interest to find cost-effective out-of-autoclave manufacturing processes without diminishing the laminate quality. The aim of this study is to evaluate the quality of fibre metal laminate panels adhesively bonded and cured using resistance heating. Three manufacturing processes are compared for different layups with an embedded steel mesh at the mid-plane: autoclave curing, resistance bonding of two (autoclave-cured) panels and complete out-of-autoclave resistance curing of panels. Interlaminar shear strength tests and optical microscopy analysis showed that resistance bonding is a promising technique, leading to results comparable to autoclave curing. Resistance curing led to an interlaminar shear strength decrease of 30–60%. A study of the correlation between degree of cure and distance from the mesh revealed the potential of resistance bonding to be used for flexible embedded mesh geometries and on-site repairs. However, it is an expensive process, especially when it comes to large parts.2123 and induction heating2426 have been investigated to cure or adhesively bond glass and carbon fibre reinforced thermoset composites. The resulting material properties were similar to those obtained with traditional manufacturing techniques, but in some cases, the presence of defects, such as the amount of voids, increased and reached content values up to 20% due to the lower pressure applied during curing.Research findings have been reported on out-of-autoclave techniques that can allow localized curing and/or bonding of thermosets. Their common goal is to reduce production costs and focus heating on specific areas. Microwave radiation2731 Those studies demonstrated the potential of resistance heating for joining composites and showed the effect of input parameters, materials and heater mesh on the quality of joints. The same concept has also been investigated to cure thermoset adhesives, resulting in high strength joints with potentially lower manufacturing costs.3234 An important aspect that has been investigated is the identification of processing windows based on input parameters, such as heating elements configuration, to accelerate the curing process with resistance heating.36Another potential localized out-of-autoclave manufacturing technique is resistance heating through the use of a metal mesh embedded at the bondline or in the laminates. This method has been employed extensively to weld thermoplastic composite parts.Using the concept of resistance heating to replace the autoclave curing process of GLARE, or to eliminate a second curing cycle when reinforcing GLARE panels, could lead to significant cost reductions. Autoclave manufacturing could be partly replaced by a less expensive, yet more adaptable equipment, consisting mainly of a vacuum bag and a power supply. This high flexibility brings new design opportunities for manufacturing innovative parts, as well as for repair applications. For instance, the location, position and shape of repair patches would be less restricted and on-site repairs using GLARE patches could be made possible. The shape of the resistance heater elements can be customizable and the temperature is generated only where it is required.The main concern is how the heating elements (or mesh) would affect the quality of the final laminate and how a uniform heating distribution can be guaranteed.Therefore, the aim of this study is to evaluate the quality of FMLs adhesively bonded or cured using resistance heating.Three different manufacturing processes are compared: (1) autoclave curing of GLARE panels, (2) resistance bonding of two autoclave-cured GLARE panels and (3) resistance curing of full GLARE panels (complete out-of-autoclave manufacturing). In order to assess the effect of the different manufacturing techniques, a detailed examination of the GLARE panels was carried out based on interlaminar shear strength (ILSS) tests and optical microscopy of the cross-sections and fracture modes.Two types of GLARE laminates were used in this study: GLARE 3-4/3-0.3 and GLARE 5-4/3-0.3. Both laminates consist of four 0.3 mm thick 2024-T3 aluminium layers, bonded together with glass fibre prepregs S2-glass/FM94. The difference between GLARE 3 and GLARE 5 laminates lies in the layup sequence of the prepregs. In GLARE 3, each glass prepreg laminate between the aluminium plates is made of uni-directional (UD) plies with a layup of [0/90]. In GLARE 5, the layup is Prior to bonding, the aluminium surfaces were pre-treated with chromic acid anodizing and primed with BR 127 .The specifications of the stainless steel heater mesh used in this study are listed in T) of P) and vacuum bag (V) pressures are maintained at 6 bar and 1 bar, respectively. In order to evaluate the effect of the steel mesh on the quality of the panels, independently of the manufacturing process, the autoclave was used to manufacture panels with and without a mesh, as schematically illustrated in The standard autoclave cycle for GLARE panels manufacturing is shown in The work presented in this paper distinguishes between resistance bonding (RB) and resistance curing (RC) of GLARE panels. In the case of the resistance bonding method, firstly, two separate GLARE panels are cured in the autoclave. Secondly, the two panels are brought together with an adhesive layer or glass prepreg layer in between. This layer is subsequently cured using resistance heating (out-of-autoclave secondary bonding), as shown in During both techniques, a voltage is applied to the metal mesh, which heats up due to its electrical resistance. By following the temperature set points given in the standard autoclave cycle see , it is pThe presence of the epoxy layers ensures electrical isolation between the heater mesh and the aluminium layers. In addition to this, the protective liner which is applied to each single aluminium layer has a very low electrical conductivity. Consequently, the chance of short circuits is reduced during manufacturing.The direct voltage (DC) was provided by the power supply and controlled manually in order to follow the temperature set point shown in Two types of GLARE panels were manufactured: (1) “Full surface mesh”-panels and (2) “Mesh stripe”-panels. In the first, the steel mesh area covers the complete surface area of the GLARE panel. For this type, panels were manufactured using the three different methods mentioned in ‘Manufacturing methods’ section. In the second one, two autoclave cured GLARE panels were bonded using only a mesh stripe, 12.5 mm wide, positioned at the centre of the panels. The aim was to assess the surface area of the embedded mesh needed to guarantee a certain degree of cure.A “Full surface mesh” panel indicates that the embedded mesh covered the entire surface area. In total, eight GLARE 3-4/3-0.3 and eight GLARE 5-4/3-0.3 panels were manufactured according to the layups listed in One “mesh stripe” panel was manufactured according to the following procedure: two GLARE 5 panels were first cured in the autoclave, then bonded using resistance heating with a 12.5 mm wide mesh stripe. The electrical current was controlled in such a way that the temperature at the surface of the panel above the mesh was between The temperature, electrical voltage and current curves were recorded during the out-of-autoclave manufacturing of GLARE panels using a full surface mesh. A representative example of the curves for resistance bonded GLARE 3 panels is shown in Representative curves for resistance cured GLARE 3 panels are shown in In order to evaluate the performance of the out-of-autoclave manufacturing methods proposed in this work, ILSS tests were performed. It is expected to provide insights into manufacturing quality and the effect of degree of cure on shear strength and adhesion of the epoxy layers.For each full surface mesh panels – listed in In the case of the mesh stripe panel, a total of six ILSS specimens in the 38 A schematic figure of the setup is given in The ILSS tests were performed according to the ASTM D2344 standard for short-beam strength of polymer matrix composite materials and their laminates.maxF is the maximum load, and W and L are the width and length of the specimen, respectively. The ILSS was calculated based on the maximum force measured in the force-displacement curves , as giveFor both GLARE 3 and GLARE 5 samples manufactured in the autoclave (A1 to A8), the heater mesh did not have a significant effect on the ILSS values, remaining within scatter range. Resistance bonded specimens (RB1 to RB4) displayed similar shear strength values to the autoclave panels, with the exception of RB2, which dropped to 47.7 MPa. This is consistent with the failure mode presented in Cross-sections of the panels manufactured according to the methods and layups presented in The quality of the outer prepreg layers for GLARE 3 specimens manufactured by resistance bonding and curing is compared on These observations can explain the failure modes witnessed in For GLARE 5 panels, the use of four pure resin layers at the mesh interface  led to cions see . The ILSThe Although also present, the change of slope and the plastic deformation in positions TC I and TC II is almost insignificant when compared with positions TC III, TC IV and TC V.As for the failure modes, positions TC I and TC II fail similarly as the specimens for full surface mesh resistance bonding using pure epoxy (RB1 and RB3): intralaminar failure in the prepreg layer close to the aluminium layer . This inThe failure mechanism was significantly different for positions TC III, TC IV and TC V. This interface failure justifies the different The average ILSS value of position TC I (1–3), where the mesh stripe was located, was 47.6 MPa. Specimens adjacent to the mesh, TC II (1–3) – 30 mm from the centre of the mesh, had similar ILSS values (50.5 MPa). At distances farther away from the mesh, the average shear strength decreases significantly at 90 mm distance by 50% (TC III) and from 150 mm on by 60% (TC IV and TC V). ILSS specimens tested in the transverse direction showed similar shear strength values as in the longitudinal direction.The ILSS values of positions TC I and TC II (47.6 to 50.9 MPa) are of the same order as the one obtained for RB3 specimens , cross-sections were observed by optical microscopy, as was the case for full surface mesh panels. These observations can justify and support the significant difference in the mechanical behaviour of the specimens close to the mesh – Positions TC I and TC II, and far from the mesh – Positions TC III, TC IV and TC V. The large voids observed in the latter confirm the poor manufacturing quality and corresponding poor mechanical performance observed at those locations.Based on the ILSS and microscopy results presented in ‘Full surface mesh panels’ section for the autoclave cured, resistance bonded and resistance cured specimens, three main observations can be highlighted.Firstly, for the autoclave cured specimens, there were minor to no changes in the quasi-static behaviour and in the cross-section quality without  and with  an embedded stainless steel mesh see  and 11. Secondly, the ILSS values, failure modes and corresponding cross-section quality were comparable for the autoclave cured and resistance bonded GLARE 3 and GLARE 5 panels. The exception which did not follow this trend was, similarly to the autoclave cured panels, the resistance bonded GLARE 3 panel with prepreg layers adjacent to the mesh (RB2 panel). It is assumed that the epoxy volume content was not sufficient to impregnate the heater mesh properly. Therefore, the crack initiated at the epoxy-heater mesh interface for RB2 specimens .Finally, the resistance curing method produced panels of distinctively lower quality with an increased presence of voids in all prepreg layers (including the ones adjacent to the heater mesh). This led to a decrease in the ILSS values and the onset of failure in the outer prepreg layers. As voids disrupt the homogeneity of the material and act as crack initiators, a higher void content consequently increases the chance of failure at lower stress values and thus, leads to a decrease of the (static) strength. However, this behaviour was more noticeable for the GLARE 5 specimens, compared to GLARE 3, as the void content was likely higher with a lower aluminium surface area over the cross-section .Using a mesh stripe instead of a full surface mesh for resistance bonding of GLARE panels severely affects the temperature distribution see . Thus, tα, can be estimated from TC I to TC V based on Kamal–Sourour’s cure kinetics model presented in Abouhamzeh et al.39 In order to do so, three main assumptions were made. Firstly, the same heating/cooling rate for all positions as the one used in the standard manufacturing cycle was assumed .As previously presented in 40 These findings suggest that using a spacing of approximately 60 mm between mesh stripes would allow to maintain reasonable degree of cure and manufacturing quality. This can provide flexibility in the case where a more complex mesh geometry might be required depending on the parts to be resistance bonded.For both cases, as the distance from the mesh increases, the degree of cure and ILSS values significantly drop between 30 mm (TC II) and 90 mm (TC III), which is consistent with literature on epoxy/glass fibre systems submitted to different cure cycles.Three manufacturing techniques for GLARE panels were investigated and compared: full autoclave curing, resistance bonding of two autoclave-cured panels and complete out-of-autoclave resistance curing. For the latter two methods, a steel mesh was placed at the panels’ mid-plane for bonding or curing through resistance heating. The effect of the heater element was investigated as a first step for autoclave cured panels. No major differences in the static behaviour and manufacturing quality were found between panels with and without an embedded heater mesh.The comparison of the different manufacturing techniques and layups with an embedded steel mesh across the whole surface at the mid-plane showed that resistance bonding is a promising technique, which leads to comparable ILSS values to the fully autoclave cured samples with a maximum decrease of 10%. Resistance cured samples, however, do not show sufficient manufacturing quality. The significant presence of voids leads to a decrease of the ILSS values, especially for the GLARE 5 samples. In all cases, the importance of a proper mesh impregnation was noted. The best quality was obtained with pure epoxy layers at the mesh interface, while the use of only one prepreg layer on each side of the mesh was more likely to promote crack initiation.As a first step toward a flexible heater mesh geometry, two GLARE 5 panels were resistance bonded using a 12.5 mm wide (stripe) heater element. The study showed that the degree of cure and ILSS values at distances larger than 30 mm from the mesh decreased significantly. This suggests that a spacing of 60 mm between mesh stripes would allow to maintain high quality and decrease energy consumption during manufacturing. Further investigation into customisable mesh dimensions for flexible on-site repairs could be a focus of future research.The promising results obtained for the resistance bonded panels with an embedded mesh across the full surface demonstrated the capability to accomplish comparable quality to autoclave manufacturing with minimal equipment . Hence, this flexible technique could eliminate a second costly autoclave cycle in the case where, for instance, doublers or stringers need to be bonded to GLARE panels. Furthermore, it can be used for assembly of larger GLARE panels through e.g. resistance bonded scarf joints.