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1. Rapid Micromolding of Sub-100 µm Microfluidic Channels Using an 8K Stereolithographic Resin 3D Printer

   https://doi.org/10.3390/mi14081519  

   Vedhanayagam, Arpith ; Golfetto, Michael ; Ram, Jeffrey L. ; Basu, Amar S. ( August 2023 , Micromachines)

   Nguyen, Nam-Trung ; Munoz, Rodrigo Alejandro ; Kalinke, Cristiane (Ed.)

   Engineering microfluidic devices relies on the ability to manufacture sub-100 micrometer fluidic channels. Conventional lithographic methods provide high resolution but require costly exposure tools and outsourcing of masks, which extends the turnaround time to several days. The desire to accelerate design/test cycles has motivated the rapid prototyping of microfluidic channels; however, many of these methods (e.g., laser cutters, craft cutters, fused deposition modeling) have feature sizes of several hundred microns or more. In this paper, we describe a 1-day process for fabricating sub-100 µm channels, leveraging a low-cost (USD 600) 8K digital light projection (DLP) 3D resin printer. The soft lithography process includes mold printing, post-treatment, and casting polydimethylsiloxane (PDMS) elastomer. The process can produce microchannels with 44 µm lateral resolution and 25 µm height, posts as small as 400 µm, aspect ratio up to 7, structures with varying z-height, integrated reservoirs for fluidic connections, and a built-in tray for casting. We discuss strategies to obtain reliable structures, prevent mold warpage, facilitate curing and removal of PDMS during molding, and recycle the solvents used in the process. To our knowledge, this is the first low-cost 3D printer that prints extruded structures that can mold sub-100 µm channels, providing a balance between resolution, turnaround time, and cost (~USD 5 for a 2 × 5 × 0.5 cm^3 chip) that will be attractive for many microfluidics labs. 

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2. Breath-synchronized electrical stimulation of the expiratory muscles in mechanically ventilated patients: a randomized controlled feasibility study and pooled analysis

   https://doi.org/10.1186/s13054-020-03352-0  

   Jonkman, Annemijn H. ; Frenzel, Tim ; McCaughey, Euan J. ; McLachlan, Angus J. ; Boswell-Ruys, Claire L. ; Collins, David W. ; Gandevia, Simon C. ; Girbes, Armand R. ; Hoiting, Oscar ; Kox, Matthijs ; et al ( December 2020 , Critical Care)

   null (Ed.)

   Abstract Background Expiratory muscle weakness leads to difficult ventilator weaning. Maintaining their activity with functional electrical stimulation (FES) may improve outcome. We studied feasibility of breath-synchronized expiratory population muscle FES in a mixed ICU population (“Holland study”) and pooled data with our previous work (“Australian study”) to estimate potential clinical effects in a larger group. Methods Holland: Patients with a contractile response to FES received active or sham expiratory muscle FES (30 min, twice daily, 5 days/week until weaned). Main endpoints were feasibility (e.g., patient recruitment, treatment compliance, stimulation intensity) and safety. Pooled: Data on respiratory muscle thickness and ventilation duration from the Holland and Australian studies were combined ( N  = 40) in order to estimate potential effect size. Plasma cytokines (day 0, 3) were analyzed to study the effects of FES on systemic inflammation. Results Holland: A total of 272 sessions were performed (active/sham: 169/103) in 20 patients ( N  = active/sham: 10/10) with a total treatment compliance rate of 91.1%. No FES-related serious adverse events were reported. Pooled: On day 3, there was a between-group difference ( N  = active/sham: 7/12) in total abdominal expiratory muscle thickness favoring the active group [treatment difference (95% confidence interval); 2.25 (0.34, 4.16) mm, P  = 0.02] but not on day 5. Plasma cytokine levels indicated that early FES did not induce systemic inflammation. Using a survival analysis approach for the total study population, median ventilation duration and ICU length of stay were 10 versus 52 ( P  = 0.07), and 12 versus 54 ( P  = 0.03) days for the active versus sham group. Median ventilation duration of patients that were successfully extubated was 8.5 [5.6–12.2] versus 10.5 [5.3–25.6] days ( P  = 0.60) for the active ( N  = 16) versus sham ( N  = 10) group, and median ICU length of stay was 10.5 [8.0–14.5] versus 14.0 [9.0–19.5] days ( P  = 0.36) for those active ( N  = 16) versus sham ( N  = 8) patients that were extubated and discharged alive from the ICU. During ICU stay, 3/20 patients died in the active group versus 8/20 in the sham group ( P  = 0.16). Conclusion Expiratory muscle FES is feasible in selected ICU patients and might be a promising technique within a respiratory muscle-protective ventilation strategy. The next step is to study the effects on weaning and ventilator liberation outcome. Trial registration: ClinicalTrials.gov, ID NCT03453944. Registered 05 March 2018—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03453944 . 

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3. Design, Fabrication, and Validation of a Petri Dish-Compatible PDMS Bioreactor for the Tensile Stimulation and Characterization of Microtissues

   https://doi.org/10.3390/mi11100892  

   Alhudaithy, Soliman ; Abdulmalik, Sama ; Kumbar, Sangamesh G. ; Hoshino, Kazunori ( October 2020 , Micromachines)

   null (Ed.)

   In this paper, we report on a novel biocompatible micromechanical bioreactor (actuator and sensor) designed for the in situ manipulation and characterization of live microtissues. The purpose of this study was to develop and validate an application-targeted sterile bioreactor that is accessible, inexpensive, adjustable, and easily fabricated. Our method relies on a simple polydimethylsiloxane (PDMS) molding technique for fabrication and is compatible with commonly-used laboratory equipment and materials. Our unique design includes a flexible thin membrane that allows for the transfer of an external actuation into the PDMS beam-based actuator and sensor placed inside a conventional 35 mm cell culture Petri dish. Through computational analysis followed by experimental testing, we demonstrated its functionality, accuracy, sensitivity, and tunable operating range. Through time-course testing, the actuator delivered strains of over 20% to biodegradable electrospun poly (D, L-lactide-co-glycolide) (PLGA) 85:15 non-aligned nanofibers (~91 µm thick). At the same time, the sensor was able to characterize time-course changes in Young’s modulus (down to 10–150 kPa), induced by an application of isopropyl alcohol (IPA). Furthermore, the actuator delivered strains of up to 4% to PDMS monolayers (~30 µm thick), simultaneously characterizing their elastic modulus up to ~2.2 MPa. The platform repeatedly applied dynamic (0.23 Hz) tensile stimuli to live Human Dermal Fibroblast (HDF) cells for 12 hours (h) and recorded the cellular reorientation towards two angle regimes, with averages of −58.85° and +56.02°. The device biocompatibility with live cells was demonstrated for one week, with no signs of cytotoxicity. We can conclude that our PDMS bioreactor is advantageous for low-cost tissue/cell culture micromanipulation studies involving mechanical actuation and characterization. Our device eliminates the need for an expensive experimental setup for cell micromanipulation, increasing the ease of live-cell manipulation studies by providing an affordable way of conducting high-throughput experiments without the need to open the Petri dish, reducing manual handling, cross-contamination, supplies, and costs. The device design, material, and methods allow the user to define the operational range based on their targeted samples/application. 

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4. Biocompatible micro tweezers for 3D hydrogel organoid array mechanical characterization

   https://doi.org/10.1371/journal.pone.0262950  

   Alhudaithy, Soliman ; Hoshino, Kazunori ( January 2022 , PLOS ONE)

   Jabbari, Esmaiel (Ed.)

   This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (μTweezers) capable of the stiffness characterization and manipulation of hydrogel-based organoids. The system showed great potential for complementing established mechanical characterization methods such as Atomic Force Microscopy (AFM), parallel plate compression (PPC), and nanoindentation, while significantly reducing the volume of valuable hydrogels used for testing. We achieved a volume reduction of ~0.22 μl/sample using the μTweezers vs. ~157 μl/sample using the PPC, while targeting high-throughput measurement of widely adopted micro-mesoscale (a few hundred μm-1500 μm) 3D cell cultures. The μTweezers applied and measured nano-millinewton forces through cantilever’ deflection with high linearity and tunability for different applications; the assembly is compatible with typical inverted optical microscopes and fit on standard tissue culture Petri dishes, allowing mechanical compression characterization of arrayed 3D hydrogel-based organoids in a high throughput manner. The average achievable output per group was 40 tests per hour, where 20 organoids and 20 reference images in one 35 mm petri dish were tested, illustrating efficient productivity to match the increasing demand on 3D organoids’ applications. The changes in stiffness of collagen I hydrogel organoids in four conditions were measured, with ovarian cancer cells (SKOV3) or without (control). The Young’s modulus of the control group (Control—day 0, E = 407± 146, n = 4) measured by PPC was used as a reference modulus, where the relative elastic compressive modulus of the other groups based on the stiffness measurements was also calculated (control-day 0, E = 407 Pa), (SKOV3-day 0, E = 318 Pa), (control-day 5, E = 528 Pa), and (SKOV3-day 5, E = 376 Pa). The SKOV3-embedded hydrogel-based organoids had more shrinkage and lowered moduli on day 0 and day 5 than controls, consistently, while SKOV3 embedded organoids increased in stiffness in a similar trend to the collagen I control from day 0 to day 5. The proposed method can contribute to the biomedical, biochemical, and regenerative engineering fields, where bulk mechanical characterization is of interest. The μTweezers will also provide attractive design and application concepts to soft membrane-micro 3D robotics, sensors, and actuators. 

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5. Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy

   Ramamurthy, R ; Zheng, WT ; George, S ; Wan, M ; Zhou, Y ; Lu, B ; Bishop, C ; Atala, A ; Porada, C ; Almeida-Porada, G ( December 2021 , ASH)

   2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures: No relevant conflicts of interest to declare. 

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