Microfluidic Chamber Research Articles

Melanoma-on-a-chip model for anticancer drug injecting delivery method

The pharmaceutical and cosmetic industries are encountering a challenge in adopting new study models for product development. there has been a growing interest in organ-on-a-chip systems, and particularly for generating skin models. While numerous alternatives replicating high-fidelity skin models exist, there is a notable absence of melanoma study's methodology specifically on these microfluidic chips. This work introduces a novel skin-on-a-chip device featuring two microfluidic chambers, facilitating a 3D cell co-culture involving fibroblasts, keratinocytes, and melanoma cells. The design of this organ-on-a-chip has enabled the administration of the anticancer treatment Gemcitabine using an injection system within the chip. The results of this work have shown a significant impact on the co-culture distribution of cells, decreasing the population of cancerous cells after the administration of Gemcitabine. The work presented in this article demonstrates the effectiveness of the chip and the administration method for testing anti-melanoma therapies and position this technology as an enhanced fidelity model for studying melanoma while providing an alternative for real-time monitoring of drug testing.

Primary sensory neuron-derived miR-let-7b underlies stress-elicited psoriasis

Psoriasis, a chronic autoimmune skin condition with significant global morbidity, badly impairs patients’ quality of life. Stress has been identified as a prominent trigger for psoriasis, and effectively management of stress can ameliorate its pathological manifestations. However, the precise mechanisms by which stress influences psoriasis remain elusive. In this study, we found that mice subjected to chronic social defeat stress (CSDS) exhibit severer imiquimod (IMQ)-induced psoriasis with increased epidermal scaling, epidermal hyperplasia, number of epidermal ridges, itch, and skin inflammation than control mice. Mechanistic study reveals that CSDS leads to an elevated release of miR-let-7b, an endogenous ligand of Toll-like receptor 7 (TLR7), from the peripheral terminal of dorsal root ganglia (DRG) neurons into the skin. This process can stimulate skin-resident macrophages to release cytokines (such as IL-6 and TNF-a) and chemokines (such as MCP-1), subsequently promoting the recruitment of additional macrophages into the skin. Notably, the specific blockade of miR-let-7b in DRG neurons effectively relieve stress-induced exacerbations of psoriasis. Furthermore, intradermal injection of synthetic miR-let-7b can induce a psoriasis-like phenotype in wildtype mice, a phenomenon that can be countered by the application of a TLR7 antagonist. Additionally, microfluidic chamber coculture assays demonstrated that miR-let-7b released by DRG neurons activates macrophages via TLR7 expressed on these immune cells. Totally, this study found that stress-induced upregulation and release of miR-let-7b from DRG neurons stimulates macrophages to secrete more inflammatory cytokines and chemokines, thereby exacerbating skin inflammation and the psoriatic phenotype. These findings provide a potential therapeutic strategy targeting the miR-let-7b/TLR7 pathway to alleviate stress-induced exacerbation of psoriasis.

Skeletal myotubes expressing ALS mutant SOD1 induce pathogenic changes, impair mitochondrial axonal transport, and trigger motoneuron death

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2 + transients and reactive oxygen species (i.e., H2O2). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes.

A New Shear-Stress-Based Point-of-Care Technology for Evaluation of the Hemostatic Pattern in Whole Blood

The currently available point-of-care hemostasis tests are burdened by criticisms concerning the use of different activators and inhibitors and the lack of dynamic flow. These operating conditions may constitute an impediment to the determination of the patient’s hemostatic condition. Hence, the diffusion of these tests in clinical practice is still limited to specific scenarios. In this work, we present a new method for analyzing the patient’s global hemostasis based on the visualization of the main components of the coagulation process and its computerized quantitative image analysis. The automated “Smart Clot” point-of-care system presents a micro-fluidic chamber in which whole blood flows, without the addition of any activator or inhibitor. In this micro-channel, platelet adhesion, activation and aggregation to the type I collagen-coated surface take place (primary hemostasis), leading to the production of endogenous thrombin on the surface of platelet aggregates and the consequent fibrin mesh and thrombus formation (secondary hemostasis). These observations are verified by inhibiting primary hemostasis with the antiplatelet drugs Indomethacin (−70% on platelet aggregation, −60% on fibrin(ogen) formation) and Tirofiban (complete inhibition of platelet aggregation and fibrin(ogen) formation) and secondary hemostasis with the antithrombin drugs Heparin (−70% on platelet aggregation, −80% on fibrin(ogen) formation) and Lepirudin (−80% on platelet aggregation, −90% on fibrin(ogen) formation). Smart Clot, through a single test, provides quantitative results concerning platelet aggregation and fibrin formation and is suitable for undergoing comparative studies with other coagulation point-of-care devices.

A microphysiological system for studying barrier health of live tissues in real time

Epithelial cells create barriers that protect many different components in the body from their external environment. Increased gut barrier permeability (leaky gut) has been linked to several chronic inflammatory diseases. Understanding the cause of leaky gut and effective interventions are elusive due to the lack of tools that maintain tissue’s physiological environment while elucidating cellular functions under various stimuli ex vivo. Here we present a microphysiological system that records real-time barrier permeability of mouse colon in a physiological environment over extended durations. The system includes a microfluidic chamber; media composition that preserves microbiome and creates necessary oxygen gradients across the barrier; and integrated sensor electrodes for acquiring transepithelial electrical resistance (TEER). Our results demonstrate that the system can maintain tissue viability for up to 72 h. The TEER sensors can distinguish levels of barrier permeability when treated with collagenase and low pH media and detect different thickness in the tissue explant.

Reusable Microfluidic Chambers for Single-Molecule Microscopy.

Maintaining a consistent environment in single-molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in nonspecific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids, or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments, as biotin can be readily incorporated into these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times by using photocleavable biotin (PC-biotin) and UV-light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5 min UV exposure of a specific wavelength. Nonoptimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality as assessed by the nonspecific binding of fluorescently labeled DNA and protein samples and the recovery of the DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.

Characterization of AI-enhanced magnetophoretic transistors operating in a tri-axial magnetic field for on-chip bioparticle sorting.

We demonstrate two general classes of magnetophoretic transistors, called the "trap" and the "repel-and-collect" transistors, capable of switching single magnetically labeled cells and magnetic particles between different paths in a microfluidic chamber. Compared with prior work on magnetophoretic transistors operating in a two-dimensional in-plane rotating field, the use of a tri-axial magnetic field has the fundamental advantages of preventing particle cluster formation and better syncing of single particles with the general operating clock. We use finite element methods to investigate the energy distribution on the chip surface and to predict the particle behavior at various device geometries. We then fabricate the proposed transistors and compare the experimental results with the simulation predictions. We found that with gate electrical currents of ~ 40mA for a transistor with proper geometry, complete switching of magnetic particles with diameters in the range of 8-15μm is achieved. We show that the device is reliable and works well at different magnetic field strengths (50-100 Oe) and frequencies (0.05-0.5Hz). We also employed an image processing code with a trained convolutional neural network to automate the proposed transistors for identifying and sorting particles with various sizes and magnetic susceptibilities with accuracies higher than 98%. The proposed transistors can be used in designing novel magnetophoretic circuits for important applications in biomedical microdevices and single-cell biology.

Emergence of synchronized growth oscillations in filamentous fungi.

Many species of soil fungi grow in the form of branched networks that enable long-range communication and mass flow of nutrient. These networks play important roles in the soil ecosystem as a major decomposer of organic materials. While there have been investigations on the branching of the fungal networks, their long-term growth dynamics in space and time is still not very well understood. In this study, we monitor the spatio-temporal growth dynamics of the plant-promoting filamentous fungus Serendipita indica for several days in a controlled environment within a microfluidic chamber. We find that S. indica cells display synchronized growth oscillations with the onset of sporulation and at a period of 3 h. Quantifying this experimental synchronization of oscillatory dynamics, we show that the synchronization can be recapitulated by the nearest neighbour Kuramoto model with a millimetre-scale cell-cell coupling. The microfluidic set-up presented in this work may aid the future characterization of the molecular mechanisms of the cell-cell communication, which could lead to biophysical approaches for controlling fungi growth and reproductive sporulation in soil and plant health management.

Mitigation of almond leaf scorch by a peptide that inhibits the motility of Xylella fastidiosa.

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that is a menace to the agriculture worldwide threating economically relevant crops such as almond. The pathogen presents a dual lifestyle in the plant xylem, consisting of sessile microbial aggregates and mobile independent cells that move by twitching motility. The latter is essential for the systemic colonization of the host and is mediated through type IV pili. In previous reports, it has been demonstrated that peptides can affect different key processes of X. fastidiosa, but their effect on motility has never been assessed. In the present work, peptides previously identified and newly designed analogs were studied for its effect in vitro on the motility of X. fastidiosa and their protective effect against almond leaf scorch was determined. By assessing the twitching fringe width in colonies and using microfluidic chambers, the inhibitory effect of BP100 on twitching motility was demonstrated. Interestingly, type IV pili of BP100-treated cells were similar in frequency and length, and presented no morphological differences when compared to the non-treated control. The application of BP100 by endotherapy in almond plants inoculated with X. fastidiosa under greenhouse conditions significantly reduced population levels and showed less affected xylem vessels, which correlated with decreased disease symptoms. Therefore, BP100 is a promising candidate to manage almond leaf scorch caused by X. fastidiosa.

Gradient-induced instability in tumour spheroids unveils the impact of microenvironmental nutrient changes

Tumours often display invasive behaviours that induce fingering, branching and fragmentation processes. The phenomenon, known as diffusional instability, is driven by differential cell proliferation, migration, and death due to the presence of metabolite and catabolite concentration gradients. An understanding of the intricate dynamics of this spatially heterogeneous process plays a key role in the investigation of tumour growth and invasion. In this study, we developed an in vitro tumour invasion assay to investigate cell invasiveness in tumour spheroids under a chemotactic stimulus. Our method, employing tumour spheroids seeded in a 3D collagen gel within a microfluidic chemotaxis chamber, focuses on the role of diffusive gradients. Using Time-Lapse Microscopy, the dynamic evolution of tumour spheroids was monitored in real-time, providing a comprehensive view of the morphological changes and cell migration patterns under different chemotactic conditions. Specifically, we explored the impact of fetal bovine serum (FBS) gradients on the behaviour of CT26 mouse colon carcinoma cells and compared the effects of varying FBS concentrations to two isotropic control conditions. Furthermore, a finite element in silico model was developed to quantify the diffusive flow of nutrients in the chemotaxis chamber and obtain a detailed understanding of tumour dynamics. Our findings reveal that the presence of a chemotactic gradient significantly influences tumour invasiveness, with higher concentrations of nutrients associated with increased cancer growth and cell migration.

Von Willebrand Factor Collagen-Binding Activity and Von Willebrand Factor-Mediated Platelet Adhesion in Patients with Coronary Artery Disease.

In this study, we investigated von Willebrand factor (VWF)-related parameters in 30 patients with stable coronary artery disease (CAD) and 50 patients without CAD. In both groups, the following were measured: the VWF antigen level (VWF:Ag); the VWF ristocetin cofactor activity (VWF:RCo); the VWF collagen-binding activity (VWF:CB); and VWF-mediated platelet adhesion. Platelet adhesion was measured in whole blood at a shear rate of 1300 s-1 using a microfluidic chamber with a collagen-coated surface. VWF:Ag and VWF:RCo were found to be the same in both groups of patients. However, VWF:CB was found to be lower in patients with CAD compared with patients without CAD, with values of 106.7% (82.1; 131.6) and 160.4% (112.5; 218.1), respectively (p < 0.001). The decrease in platelet adhesion after GPIb inhibition was more pronounced in patients with CAD compared with patients of the control group, with recorded values of 76.0% (60.6; 82.1) and 29.3% (0.0; 60.4), respectively (p < 0.001). After adjusting for traditional risk factors, the odds ratio for CAD was found to be 0.98 (95% CI, 0.97-0.99; p = 0.011) per 1% increase in VWF:CB activity, and 1.06 (95% CI, 1.03-1.09; p < 0.001) per 1% decrease in GPIb-mediated platelet adhesion. The findings presented in this paper indicate a possible critical role played by complex VWF-collagen-platelet interactions in the development of CAD.

Functionalized monodisperse microbubble production: microfluidic method for fast, controlled, and automated removal of excess coating material

Functionalized monodisperse microbubbles have the potential to boost the sensitivity and efficacy of molecular ultrasound imaging and targeted drug delivery using bubbles and ultrasound. Monodisperse bubbles can be produced in a microfluidic flow focusing device. However, their functionalization and sequential use require removal of the excess lipids from the bubble suspension to minimize the use of expensive ligands and to avoid competitive binding and blocking of the receptor molecules. To date, excess lipid removal is performed by centrifugation, which is labor intensive and challenging to automate. More importantly, as we show, the increased hydrostatic pressure during centrifugation can reduce bubble monodispersity. Here, we introduce a novel automated microfluidic ’washing’ method. First, bubbles are injected in a microfluidic chamber 1 mm in height where they are left to float against the top wall. Second, lipid-free medium is pumped through the chamber to remove excess lipids while the bubbles remain located at the top wall. Third, the washed bubbles are resuspended and removed from the device into a collection vial. We demonstrate that the present method can (i) reduce the excess lipid concentration by 4 orders of magnitude, (ii) be fully automated, and (iii) be performed in minutes while the size distribution, functionality, and acoustic response of the bubbles remain unaffected. Thus, the presented method is a gateway to the fully automated production of functionalized monodisperse microbubbles.

AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro.

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

Albumin reprograms the B cell transcriptional landscape and improves neutrophil antimicrobial function in patients with decompensated cirrhosis

Background&AimPatients with acutely decompensated (AD) cirrhosis are immunocompromised and particularly susceptible to infections. This study investigated the immunomodulatory actions of albumin by which this protein may lower the incidence of infections. MethodsBlood immunophenotyping was performed in 11 AD patients and 10 healthy volunteers (HV). Bulk and single-cell (sc) RNA sequencing (RNA-seq) and flow cytometry were performed in peripheral blood mononuclear cells (PBMCs) from 20 AD patients and 34 HV exposed to albumin. Albumin’s effects on degranulation, phagocytosis, chemotaxis, and swarming of neutrophils from 6 AD patients and 9 HV were assessed by myeloperoxidase enzymatic activity, engulfment of fluorescent-labeled Escherichia coli and zymosan and interactions at single-cell resolution of neutrophils with Candida albicans in microfluidic chambers, respectively. Whole blood RNA-seq analyses were performed in 45 patients admitted for severe AD of whom 30 received albumin during hospitalization. ResultsCompared to HV, AD patients showed severe lymphopenia and defective neutrophil anti-microbial function. Bulk and scRNA-seq analyses revealed significantly (false discovery rate (FDR)<0.05) increased signatures related to B cells, myeloid cells and CD4+ T cells in PBMCs incubated with albumin. Changes in the B cell population were confirmed by flow cytometry. Neutrophils exposed to albumin also exhibited augmented chemotactic and degranulation responses, enhanced phagocytosis, and increased pathogen-restrictive swarming. Analysis of RNA-seq data in patients who had received albumin revealed specific upregulation of signatures related to B cells and neutrophils together with transcriptional changes in CD4+ T cells (FDR<0.05). ConclusionsThe finding that albumin promotes the transcriptional reprogramming and expansion of the B cell compartment and improves neutrophil antimicrobial functions indicates mechanisms that may lower the incidence of infections in patients with severe AD receiving albumin therapy.

Atmospheric Pressure Plasma Functionalization of Sealed PDMS Microfluidics: Application to Capillary Pumping and Enhanced Cell Growth.

Microfluidic devices serve as essential tools across diverse fields like medicine, biotechnology, and chemistry, enabling advancements in analytical techniques, point-of-care diagnostics, microfluidic cell cultures, and organ-on-chip models. While polymeric microfluidics are favoured for their cost-effectiveness and ease of fabrication, their inherent hydrophobic properties necessitate surface functionalization, often post-sealing. Here, we introduce a versatile apparatus for functionalizing sealed microfluidic devices using atmospheric plasma processing, with a focus on PDMS (polydimethylsiloxane) microfluidics. Through meticulous analysis of surface properties and capillary speed, before and after plasma treatment, along with a comparison between vacuum and atmospheric plasma functionalization methods, we demonstrate the efficacy of our approach. Subsequent experimentation within 3D PDMS microfluidic chambers, combining atmospheric pressure plasma treatment with collagen coating to facilitate mesenchymal stem cells (MSCs) growth over five days, reveals enhanced initial cell adhesion and proliferation, highlighting the potential of our method for improving cell-based applications within microfluidic systems.

Fabrication of liquid-filled zoom compound eyes with a tunable focal length

The conventional bionic compound eye system encounters challenges associated with a complex zoom structure, susceptibility to wear, and elevated costs. In this paper, a preparation method of liquid-filled zoom compound eyes is proposed. A polydimethylsiloxane (PDMS) film is integrated with a microfluidic chamber composed of polymethyl methacrylate (PMMA). The microlens array (MLA, hexagonal array, diameter 800 µm, center distance 850 µm) on the PDMS film is prepared by a wettability-guided dipping method. The sub-eye focal length on zoom compound eyes (ZCEs) is 1.42 mm. By controlling the volume of deionized (DI) water injected into the chamber from 2.41 to 2.71cm3, the focal length of the main lens is adjusted from infinity to 55.51 mm, and the FOV is adjusted from 32° to 58°. This large aperture ZCE combines the advantages of monocular and compound eyes. This method has great potential for advanced micro-optical devices with a wide field of view and tunable imaging capabilities.

Evaluation of Electrochemical Sensor Using Microfluidic System

This paper presents the characterization and testing of electrochemical sensor using microfluidic system. Various geometric patterns were laser cut into the platinum working electrode of a biosensor. In this work, a microfluidic chamber was designed that allows phosphate buffer solution (PBS) to flow across the sensor, using a peristaltic pump, while varying the concentration of hydrogen peroxide. The amperometric characterization of the electrochemical sensor with 25, 50, 75, and 100[Formula: see text][Formula: see text]m perforation and 75[Formula: see text][Formula: see text]m spacing showed the highest sensitivity. This result was to be expected the purpose of patterning the sensors was to provide a 3-dimensional structure to the planar electrode in order for the enzyme, glucose oxidase, to be immobilized. Future work will include selecting one of the patterns for immobilization of glucose oxidase allowing us to realize a fully functional glucose sensor.

Boosting Microfluidic Enzymatic Cascade Reactions with pH-Responsive Polymersomes by Spatio-Chemical Activity Control.

Microfluidic flow reactors permit the implementation of sensitive biocatalysts in polymeric environments (e.g., hydrogel dots), mimicking nature through the use of diverse microstructures within defined confinements. However, establishing complex hybrid structures to mimic biological processes and functions under continuous flow with optimal utilization of all components involved in the reaction process represents a significant scientific challenge. To achieve spatial, chemical, and temporal control for any microfluidic application, compartmentalization is required, as well as the unification of different sensitive compartments in the reaction chamber for the microfluidic flow design. This study presents a self-regulating microfluidic system fabricated by a sequential photostructuring process with an intermediate chemical process step to realize pH-sensitive hybrid structures for the fabrication of a microfluidic double chamber reactor for controlled enzymatic cascade reaction (ECR). The key point is the adaptation and retention of the function of pH-responsive horseradish peroxidase-loaded polymersomes in a microfluidic chip under continuous flow. ECR is successfully triggered and controlled by an interplay between glucose oxidase-converted glucose, the membrane state of pH-responsive polymersomes, and other parameters (e.g., flow rate and fluid composition). This study establishes a promising noninvasive regulatory platform for extended spatio-chemical control of current and future ECR and other cascade reaction systems.

Characterization of 3D oxygen concentrations in microfluidic systems combining astigmatic particle tracking with phosphorescence decay measurements

In this work, we combine astigmatic particle tracking with phosphorescence decay measurements to determine 3D-resolved oxygen concentration in microfluidic systems. In our experiments we achieve uncertainties in the out-of-plane position of less than 1 µm and uncertainties in oxygen concentration of less than 2 ppm. The calibrated measurement range covers oxygen concentrations of 0.6 ppm to 27.6 ppm. A method is presented to correct for measurement errors caused by photobleaching. For this, the excitation energy, the cumulative exposure time and the spatially varying intensity profile of the laser are taken into account in the correction. With this method, low measurement errors of less than 2ppm at ambient oxygen levels can be achieved even after thousands of excitation cycles. Hence, 3D oxygen concentrations are measured in an agarose hydrogel filled microfluidic chamber across which different pressure and oxygen gradients can be set independently. The results show that oxygen diffusion is superposed by an interstitial flow, which significantly alters the resulting oxygen concentration.

P-108 Automated sperm selection software (SiD) provides similar results as human selection for ICSI

Abstract Study question Automated sperm selection or human eye-based sperm selection, does it make a difference on IVF lab outcomes and clinical outcomes? Summary answer Single sperm selection by the AI SiD was as proficient as highly experienced embryologists and embryo development may have improved for inexperienced embryologists What is known already The implementation of artificial intelligence in reproductive medicine has proven effective for a variety of applications. One application for this technology is single sperm selection during ICSI. The computer assisted program SiD, was designed to grade sperm, in real-time, based on motility parameters transforming qualitative sperm assessments into a quantitative score. SiD’s assistance can be used to select the most optimal sperm for ICSI. The application of sperm selection by AI is expected to bypass user bias, fatigue, limited training, while also optimising time. However, there are limited studies correlating the usage of AI for sperm selection with ICSI outcomes. Study design, size, duration 1 year period 608 sibling MII oocytes were randomly divided in two groups; 1-ICSI group (n = 301): ICSI performed with sperm selected by the embryologist and 2- SID-ICSI group (n = 307): ICSI performed with sperm selected using SiD software. Priority was given to sperm classified as “Best” by SID software for ICSI.. Morphology selection was performed before ICSI in both groups. Embryos were cultured during 6 days in the same conditions. Participants/materials, setting, methods Sperm preparation was performed using a microfluidic chamber (Zymot™). Sperm selection was performed as previously described, and ICSI was performed following routine procedures. Biological and clinical outcomes were analysed in both groups and compared including fertilization rate, cleavage rate, embryo and blastocyst development rate, top quality embryo rate, blastocyst development rate and pregnancy rates. Biological outcomes in both groups were also compared based on embryologist seniority. Embryo morphokinetics parameters were analysed using time-lapse system. Main results and the role of chance There was a non significant trend towards better outcomes in the SID-ICSI group for all biological outcomes including fertilization rate (83,1% vs 82,4%), cleavage rate (97,6% vs 97,2%), day 3 embryo development rate (72,9% vs 70,6%), blastocyst development rate at day 5 (49,0% vs 44,8 %), good quality blastocyst development rate at day 5 (45,1% vs 41,5%), and top quality blastocyst development rate at day 5 (25,9% vs 22,2%). Similarly, a non-significant increase was observed in all these outcomes when sperm selection is performed by a junior embryologist. Embryo development was monitored using a timelapse system. Our data show that 2nd polar body extrusion (tPB2) (4,0h vs 4,8 h [p = 0,005]) and early cleavage (t2) (27,3h vs 28,7h [p = 0,05]) happen significantly earlier when SiD is used for ICSI. No significant difference was observed for all other fertilization events (cytoplasmic wave,tPN1,tPN2,presence of cytoplasmic halo,tPNf,and cytoplasmic halo disappearance), and other cleavage timings (t3-&amp;gt;t10,tM,tSC,tEC,tSB), and embryonic cell cycles (ECC1,ECC2,ECC3,s2,s3). Euploid rate obtained with in the 2 groups was comparable (53,3% in SID-ICSI vs 44,4% in ICSI). Although there was a trend toward higher cumulative early and clinical pregnancy rate after ICSI-SID, the difference did not reach significance (43,1%vs34,0% and 27,5%vs21,2%, respectively). Limitations, reasons for caution The ZymotTM microfluidic chamber was used for sperm preparation. This device optimises sperm selection, giving rise to an already optimised population of spermatozoa. Future investigations evaluating the effect of SiD automated sperm selection software on biological outcomes in samples prepared with a less effective technique would potentially yield conversing results. Wider implications of the findings This pilot study demonstrated that the usage of the automated sperm selection software SiD gives rise to similar biological outcomes and embryo morphokinetics, indicating its effectiveness in sperm selection. We expect this program would be useful in the presence of less experienced laboratory members helping by standardizing sperm selection. Trial registration number NA