Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.

Clonal cell lines harboring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 6-8 weeks.

Prosthesis-prosthesis anastomosis using barbed sutures compared to conventional sutures under high, long-term pressure; in vitro extracorporeal circulation setup.

BACKGROUND: While barbed sutures have been extensively utilized in other disciplines, they have not been widely adopted in cardiac surgery. The lack of safety and feasibility data has limited its use within the field. To aide in the further understanding of how cardiac surgeons can use barbed sutures, we sought to develop a high-pressure in vitro simulation model. We compared knotless barbed sutures in a highly pressurized anastomosis to conventional sutures. METHODS: Ten specimens in total were utilized in prosthesis anastomosis, using 34 mm Gelweave Plexus (Terumo Aortic, Sunrise, FL 33325, USA) and 34 mm Hemabridge (Intergard Woven Hemabridge, Getinge, Göteborg, Sweden). Five models of size 3-0 barbed suture anastomoses using non-absorbable, barbed, self-retaining, monofilament polypropylene sutures (Filbloc® 3-0, Assut Europe, Rome, Italy) were compared against five conventional anastomoses using size 4-0 polypropylene monofilament (Ethicon, USA). The systems were connected using a novel-designed extracorporeal circulation system. Pressure was rapidly increased in the specimen to a mean pressure of 300-350 mmHg, running then for a minimum of 48 hours to assess anastomosis strength and endurance. RESULTS: No anastomotic dehiscence or rupture was recorded. Complex, angular anastomosis required extra stitch leakage sutures in both conventional and barbed suture specimens. CONCLUSION: Using knotless barbed sutures with an additional self-locking maneuver for prosthesis-prosthesis anastomosis in cardiac surgery is feasible in an in vitro model under long term, high-mean pressure when compared to conventional sutures. In vivo trials should be performed to further validate the in vitro findings.

Feasibility of Robotic Mitral Valve Repair Using Barbed Nonabsorbable Sutures: A Preliminary Single-Center Experience.

OBJECTIVE: Barbed nonabsorbable sutures have been widely adopted for tissue closure in noncardiac robotic surgery to improve intraoperative efficiency. Here, we examine the profile in robotic mitral valve repair (rMVR), which utilized barbed nonabsorbable sutures. To our knowledge, this is the first report to describe clinical outcomes for rMVR with barbed nonabsorbable sutures. METHODS: A retrospective review identified 90 patients who underwent rMVR using barbed nonabsorbable sutures at our center between 2019 and 2021. The primary outcome measure was dehiscence, while other relevant outcomes included 30-day readmission and 30-day mortality. RESULTS: In addition to fixation of the mitral annuloplasty band, barbed nonabsorbable sutures were employed commonly in concomitant pericardiectomy closure (100.0%, 90 of 90), atriotomy closure (100.0%, 90 of 90), and left atrial appendage closure (if eligible; 98.8%, 83 of 84). One patient who underwent mitral valve annuloplasty using only barbed nonabsorbable suture required reoperation for annuloplasty ring dehiscence. Immediate postoperative ring dehiscence was not observed in any patients after the routine reinforcement of barbed nonabsorbable sutures with everting pledgeted polyester sutures, and no additional patients required reoperation for suture-related complications. Clinical signs of dehiscence were not observed after pericardiectomy, atriotomy, or left atrial appendage closure with barbed nonabsorbable sutures. The 30-day readmission rate was 3.3% (3 of 90), and 30-day mortality was 0% (0 of 90). CONCLUSIONS: These data suggest the initial feasibility of barbed nonabsorbable sutures in robotic cardiac surgery, specifically within rMVR. Further research is necessary to explore the long-term safety and efficacy profile of such approach.

Endoaortic Balloon Occlusion in Totally Endoscopic and Percutaneous Robotic Cardiac Surgery.

Previous studies have shown that the endoaortic balloon occlusion (EABO) can provide satisfactory aortic cross-clamping with comparable surgical outcomes to thoracic aortic clamping in the setting of minimally invasive and robotic cardiac surgery. We described our approach to EABO use in totally endoscopic and percutaneous robotic mitral valve surgery. Preoperative computed tomography angiography is required to evaluate the quality and size of the ascending aorta, identify access sites for peripheral cannulation and endoaortic balloon insertion, and screen for other vascular anomalies. Continuous bilateral upper extremity arterial pressure and cranial near-infrared spectroscopy monitoring are essential to detect obstruction of the innominate artery due to distal balloon migration. Transesophageal echocardiography is needed for continuous monitoring of balloon positioning and antegrade cardioplegia delivery. Direct fluorescent visualization of the endoaortic balloon on the robotic camera allows for verification of balloon and efficient repositioning if needed. The surgeon should assess hemodynamic and imaging information simultaneously during the balloon inflation and delivery of antegrade cardioplegia. Aortic root pressure, systemic blood pressure, and balloon catheter tension affect the position of the inflated endoaortic balloon in the ascending aorta. The surgeon should eliminate all slack in the balloon catheter and lock it into position to prevent proximal balloon migration after the completion of antegrade cardioplegia. Using scrupulous preoperative imaging assessment and continuous intraoperative monitoring, the EABO can achieve adequate cardiac arrest in totally endoscopic robotic cardiac surgery, even in patients with previous sternotomy without compromise of surgical outcomes.

An alternate approach: Percutaneous axillary cannulation for minimally invasive cardiac surgery.

BACKGROUND: Percutaneous axillary artery cannulation for cardiopulmonary bypass (CPB) offers a novel alternate approach to mechanical circulatory support for patients with contraindications to femoral perfusion. To our knowledge, this has not yet been reported in minimally invasive cardiac surgery (MICS). AIM: We aim to highlight our experience using percutaneous axillary artery cannulation to safely facilitate CPB for minimally invasive cardiac surgery MICS. METHODS: Four patients who underwent robotic cardiac surgery utilizing the axillary artery for percutaneous cannulation between November 2019 and August 2021 at a single center were identified and included in the analysis. Preoperative, intraoperative, and postoperative data were collected and analyzed to support this case series. RESULTS: There were no perioperative hematomas, brachial plexus injuries, or neurovascular injuries. Within 30-days postoperatively there was no mortality, vessel injury, stroke, new onset atrial fibrillation, or other life-threatening bleeding. CONCLUSION: Percutaneous cannulation of the axillary artery is a novel and promising CPB modality for robotic cardiac surgery in patients with extensive peripheral and aortic atherosclerotic disease.

Engineering bioactive nanoparticles to rejuvenate vascular progenitor cells.

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including type-2 diabetes mellitus, hypertension, and cardiovascular disease. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). Although several approaches have been previously explored to restore endothelial function, their widespread adoption remains tampered by systemic side effects of adjuvant drugs and unintended immune response of gene therapies. Here, we report a strategy to rejuvenate circulating vascular progenitor cells by conjugation of drug-loaded liposomal nanoparticles directly to the surface of GDM-exposed ECFCs (GDM-ECFCs). Bioactive nanoparticles can be robustly conjugated to the surface of ECFCs without altering cell viability and key progenitor phenotypes. Moreover, controlled delivery of therapeutic drugs to GDM-ECFCs is able to normalize transgelin (TAGLN) expression and improve cell migration, which is a critical key step in establishing functional vascular networks. More importantly, sustained pseudo-autocrine stimulation with bioactive nanoparticles is able to improve in vitro and in vivo vasculogenesis of GDM-ECFCs. Collectively, these findings highlight a simple, yet promising strategy to rejuvenate GDM-ECFCs and improve their therapeutic potential. Promising results from this study warrant future investigations on the prospect of the proposed strategy to improve dysfunctional vascular progenitor cells in the context of other chronic diseases, which has broad implications for addressing various cardiovascular complications, as well as advancing tissue repair and regenerative medicine.