High-Density Microporous Drainage-Integrating Sheath Flow Generator for Streamlining Microfluidic Cell Sorting Systems.

Tremendous efforts have been made to develop practical and efficient microfluidic cell and particle sorting systems; however, there are technological limitations in terms of system complexity and low operability. Here, we propose a sheath flow generator that can dramatically simplify operational procedures and enhance the usability of microfluidic cell sorters. The device utilizes an embedded polydimethylsiloxane (PDMS) sponge with interconnected micropores, which is in direct contact with microchannels and seamlessly integrated into the microfluidic platform. The high-density micropores on the sponge surface facilitated fluid drainage, and the drained fluid was used as the sheath flow for downstream cell sorting processes. To fabricate the integrated device, a new process for sponge-embedded substrates was developed through the accumulation, incorporation, and dissolution of PMMA microparticles as sacrificial porogens. The effects of the microchannel geometry and flow velocity on the sheath flow generation were investigated. Furthermore, an asymmetric lattice-shaped microchannel network for cell/particle sorting was connected to the sheath flow generator in series, and the sorting performances of model particles, blood cells, and spiked tumor cells were investigated. The sheath flow generation technique developed in this study is expected to streamline conventional microfluidic cell-sorting systems as it dramatically improves versatility and operability.

Clinical Anatomy of the Superficial Preprostatic Vein and Accessory Pudendal Artery in Robot-Assisted Radical Prostatectomy.

Purpose: We herein describe the superficial preprostatic vein (SPV) anatomy and determine its relationship with the accessory pudendal artery (APA). Materials and Methods: We reviewed 500 patients with localized prostate cancer who underwent conventional robot-assisted radical prostatectomy between April 2019 and March 2023 at our institution. SPV was defined as "any vein coming from the space between the puboprostatic ligaments and running within the retropubic adipose tissue anterior to the prostate toward the vesical venous plexus or pelvic side wall." While APA was defined as "any artery located in the periprostatic region running parallel to the dorsal vascular complex and extending caudal toward the anterior perineum." The intraoperative anatomy of each SPV and APA was described. Results: SPVs had a prevalence rate of 88%. They were preserved in 252 men (58%) and classified as I-, reversed-Y (rY)-, Y-, or H-shaped (64%, 22%, 12%, and 2%, respectively) based on their intraoperative appearance. Overall, 214 APAs were found in 142 of the 252 men with preserved SPV (56%; 165 lateral and 50 apical APAs in 111 and 41 men, respectively). SPVs were pulsatile in 39% men perhaps due to an accompanying tiny artery functioning as a median APA. Pulsations seemed to be initially absent in most SPVs but become apparent late during surgery possibly due to increased arterial and venous blood flow after prostate removal. Pulsations were common in men with ≥1 APA. Conclusions: This study, which described the anatomical variations in arteries and veins around the prostrate and their preservation techniques, revealed that preserving this vasculature may help preserve postprostatectomy erection. ClinicalTrials: The Clinical Research Registration Number is 230523D.

Inguinal hernia leads to worse immediate urinary continence after robot-assisted radical prostatectomy.

OBJECTIVES: Patients with inguinal hernia (IH) may have voiding dysfunction and weak urethra-stabilizing periurethral fascial tissues, contributing to urinary incontinence. This study aimed to review the association between IH and urinary continence after robotic-assisted radical prostatectomy (RARP). METHODS: This single-institution retrospective study included 251 consecutive cases of RARP between April 2019 and June 2022. Patients with concurrent IH or a history of adult IH repair were examined. The urine loss rate (ULR), defined as 24-h urine loss volume divided by the total urine volume immediately after urinary catheter removal (i.e., 6 or 7 postoperative days), was compared between the groups with (n = 33) and without IH (n = 214). Possible contributing factors for ULR were assessed, including age, body mass index (BMI), after benign prostatic hyperplasia surgery, prostate weight, and nerve-sparing. ULR was compared intergroup after propensity score matching countering selection biases. RESULTS: Patients with IH were older (71.3 versus. 66.8 years, p < 0.01), had lower BMI (22.8 versus. 24.3, p < 0.01), and had higher ULR (14.5% versus. 5.1%, p < 0.01). In a multiple linear regression analysis (adjusted R2 = 0.084), IH (p < 0.01) was an independent contributing factor for ULR besides advancing age (p < 0.03). After propensity score matching adjusted for patient's age and nerve-sparing, patients with IH had higher ULR (14.1% versus. 5.7%, p < 0.03) as well. CONCLUSIONS: This study first reported that IH may be one of the risk factors of urinary incontinence after RARP.

Process simplification and structure design of parallelized microslit isolator for physical property-based capture of tumor cells.

Numerous attempts have been made to develop efficient systems to purify trace amounts of circulating tumor cells (CTCs) from blood samples. However, current technologies are limited by complexities in device fabrication, system design, and process operability. Here we describe a facile, scalable, and highly efficient approach to physically capturing CTCs using a rationally designed microfluidic isolator with an array of microslit channels. The wide but thin microslit channels with a depth of several micrometers selectively capture CTCs, which are larger and less deformable than other blood cells, while allowing other blood cells to just flow through. We investigated in detail the effects of the microchannel geometry and operating parameters on the capture efficiency and selectivity of several types of cultured tumor cells spiked in blood samples as the CTC model. Additionally, in situ post-capture staining of the captured cells was demonstrated to investigate the system's applicability to clinical cancer diagnosis. The presented approach is simple in operation but significantly effective in capturing specific cells and hence it may have great potential in implementating cell physics-based CTC isolation techniques for cancer liquid biopsy.

Micropassage-embedding composite hydrogel fibers enable quantitative evaluation of cancer cell invasion under 3D coculture conditions.

Cell migration and invasion are of significant importance in physiological phenomena, including wound healing and cancer metastasis. Here we propose a new system for quantitatively evaluating cancer cell invasion in a three-dimensional (3D), in vivo tissue-like environment. This system uses composite hydrogel microfibers whose cross section has a relatively soft micropassage region and that were prepared using a multilayered microfluidic device; cancer cells are encapsulated in the core and fibroblasts are seeded in the shell regions surrounding the core. Cancer cell proliferation is guided through the micropassage because of the physical restriction imposed by the surrounding solid shell regions. Quantitative analysis of cancer cell invasion is possible simply by counting the cancer cell colonies that form outside the fiber. This platform enables the evaluation of anticancer drug efficacy (cisplatin, paclitaxel, and 5-fluorouracil) based on the degree of invasion and the gene expression of cancer cells (A549 cells) with or without the presence of fibroblasts (NIH-3T3 cells). The presented hydrogel fiber-based migration assays could be useful for studying cell behaviors under 3D coculture conditions and for drug screening and evaluation.

Development of a perfusable 3D liver cell cultivation system via bundling-up assembly of cell-laden microfibers.

Although the reconstruction of functional 3D liver tissue models in vitro presents numerous challenges, it is in great demand for drug development, regenerative medicine, and physiological studies. Here we propose a new approach to perform perfusion cultivation of liver cells by assembling cell-laden hydrogel microfibers. HepG2 cells were densely packed into the core of sandwich-type anisotropic microfibers, which were produced using microfluidic devices. The obtained microfibers were bundled up and packed into a perfusion chamber, and perfusion cultivation was performed. We evaluated cell viability and functions, and also monitored the oxygen consumption. Furthermore, fibers covered with vascular endothelial cells were united during the perfusion culture, to form vascular network-like conduits between fibers. The presented technique can structurally mimic the hepatic lobule in vivo and could prove to be a useful model for various biomedical research applications.

[A CASE OF BILATERAL SYNCHRONOUS TESTICULAR TUMORS, WHICH WAS DIFFICULT TO DIFFERENTIATE FROM MALIGNANT LYMPHOMA].

We report a case of bilateral synchronous testicular tumors, which was difficult to differentiate from malignant lymphoma. A 56-year-old man with a left scrotal mass was referred to our hospital. Ultrasonography revealed a uniformly hypoechoic mass in bilateral testes. Magnetic resonance imaging also revealed a homogeneously low-intensity lesion in the bilateral testes on T2-weighted images. Abdominal and chest computed tomography showed no lymphadenopathy or metastasis. The image findings at that time suggested a malignant lymphoma, and consequently, we performed a right radical orchiectomy. Histopathological examination revealed typical seminoma in the right testis; following this observation, left radical orchiectomy was performed, and the patient was diagnosed with synchronous bilateral testicular germ cell tumors. No recurrence or metastasis has been detected postoperatively. We recommend that the diagnosis of bilateral testicular tumors be made on the basis of patients' age, tumor marker level, and image findings.

[A CASE OF PELVIC FRACTURE URETHRAL INJURY REPAIRED BY FOURTH URETHROPLASTY].

Pelvic fracture urethral injury (PFUI) is relatively rare injury, and delayed urethroplasty is the gold standard for its repair. Because a failed urethroplasty results in increased scarring and reduced length of the available urethral segment, the salvage urethroplasty is a urologic challenge. We herein report the case of male patient with PFUI who could be salvaged after three failed urethroplasties. A 22-year-old male who had received a PFUI when he was 4 years old was referred for salvage urethroplasty after two perineal anastomotic urethroplasties and one transpubic urethroplasty had failed, leaving him unable to void and forced to keep having a suprapubic tube placed. The meatus was iatrogenically obliterated due to repeated treatments. Through an incision from the scrotum and right side of the root of the penis to the lower abdomen, the anterior urethra was circumferentially mobilized and transected at the distal urethral stump. The proximal urethral end was identified by palpitation with a metallic sound inserted through the suprapubic tube tract. The covering fibrotic scar tissue was completely removed and 8 anastomotic 4-0 polydioxanone sutures were placed to reapproximate the urethral mucosa. A pedicled gracilis muscle flap was created and wrapped around the anastomosis site and intrapelvic segment of the mobilized anterior urethra. A urethrogram 8 months after urethroplasty showed a wide caliber at the anastomosis site and a ventral midline meatotomy was subsequently performed. He is currently free from any instruments and can void well without the need for further intervention.

Direct Observation of Splitting in Oil-In-Water-In-Oil Emulsion Droplets via a Microchannel Mimicking Membrane Pores.

Direct observation of double emulsion droplet permeation through a microchannel that mimicked 100 µm membrane pores with a porosity of 66.7% provided insights regarding splitting mechanisms in porous membranes. The microchannel was fabricated by standard soft lithography, and the oil-in-water-in-oil double emulsion droplets were prepared with a glass capillary device. By changing the flow rate from 0.5 to 5.0 × 10-2 m s-1, three characteristic behaviors were observed: (a) passage into one channel without splitting; (b) division into two smaller components; and (c) stripping of the middle water phase of the double emulsion droplets into a smaller double emulsion droplet and a smaller water-in-oil single emulsion droplet. The mechanisms are discussed with respect to the balance of viscous forces and interfacial tension, the contact point with the tip of the channel, and the relative position of the innermost droplet within the middle droplet.

Collagen Microparticle-Mediated 3D Cell Organization: A Facile Route to Bottom-up Engineering of Thick and Porous Tissues.

In closely packed artificial 3D cellular constructs, cells located near the center of the constructs are not functional because of the limited supply of oxygen and nutrition. Here we describe a simple, unique, and highly versatile approach to organizing cells into thick but porous 3D tissues, using cell-sized collagen microparticles as particulate scaffolds. When cells and particles are mixed and seeded in a noncell-adhesive planar chamber, they gather to form sheet-shaped structures with a thickness of 100-150 µm. In the construct, uniformly distributed particles work as a binder between cells and modulate the strong intercellular contraction. We confirmed that several factors, including the particle/cell ratio and particle size, critically affect the stability and shrink behaviors of porous tissues prepared using mouse embryonic fibroblasts (NIH-3T3 cells). Cross-sectional observation, together with cell proliferation and viability assays, revealed that the cells composing the tissues are functional primarily because interior pores between cells/particles worked as a path for efficient molecular transport. Furthermore, we prepared thick cell tissues of a liver model using human hepatocarcinoma cells (HepG2 cells), and confirmed that liver-specific functions were upregulated when composite tissues were formed using collagen microparticles prepared with several different stabilization protocols by glutaraldehyde, genipin, and methyl acetate). The process presented would be highly useful in enabling one-step production of thick cellular constructs in which porosity and morphology are tunable.

Enhancement of osteoblastic differentiation in alginate gel beads with bioactive octacalcium phosphate particles.

The present study investigated whether alginate (Alg) hydrogel microbeads have a role in maintaining mouse bone marrow stromal ST-2 cells and release the cells after being stimulated by synthetic octacalcium phosphate (OCP), which is a mineral crystal capable of stimulating osteoblastic differentiation during a conversion process to hydroxyapatite (HA). The ST-2 cell suspension in the alginate solution, which contained various concentrations of OCP granules with diameters less than 53 µm, was extruded drop-wise into a stirred gelation solution containing BaCl2 using an encapsulator with nitrogen gas stream. The Alg-microbeads (Alg/OCP · ST-2 microbeads) that were generated, which had a diameter of approximately 400 µm, were incubated for up to 14 d and then assessed for osteoblastic differentiation. Alg-microbeads with cells were also incubated to identify the possible conversion from OCP to HA. Osteoblast differentiation markers in ST-2 cells, alkaline phosphatase (ALP) and collagen type I, were up-regulated in the presence of higher amounts of OCP. X-ray diffraction analysis and Fourier transform infrared spectroscopy confirmed that the OCP tended to convert to HA over time, suggesting that the OCP in Alg-microbeads interacts three-dimensionally with ST-2 cells and stimulates its osteoblastic differentiation. The release of ST-2 cells from the microbeads was also estimated. ST-2 cells were identified outside of the microbeads, although the cell number tended to decrease with increasing OCP. These results suggest that Alg/OCP microbeads could be used as a vehicle to activate osteoblastic cells and deliver them to sites where bone regeneration is needed.

Cell-sized condensed collagen microparticles for preparing microengineered composite spheroids of primary hepatocytes.

The reconstitution of extracellular matrix (ECM) components in three-dimensional (3D) cell culture environments with microscale precision is a challenging issue. ECM microparticles would potentially be useful as solid particulate scaffolds that can be incorporated into 3D cellular constructs, but technologies for transforming ECM proteins into cell-sized stable particles are currently lacking. Here, we describe new processes to produce highly condensed collagen microparticles by means of droplet microfluidics or membrane emulsification. Droplets of an aqueous solution of type I collagen were formed in a continuous phase of polar organic solvent followed by rapid dissolution of water molecules into the continuous phase because the droplets were in a non-equilibrium state. We obtained highly unique, disc-shaped condensed collagen microparticles with a final collagen concentration above 10% and examined factors affecting particle size and morphology. After testing the cell-adhesion properties on the collagen microparticles, composite multicellular spheroids comprising the particles and primary rat hepatocytes were formed using microfabricated hydrogel chambers. We found that the ratio of the cells and particles is critical in terms of improvement of hepatic functions in the composite spheroids. The presented methodology for incorporating particulate-form ECM components in multicellular spheroids would be advantageous because of the biochemical similarity with the microenvironments in vivo.

Patterned hydrogel microfibers prepared using multilayered microfluidic devices for guiding network formation of neural cells.

Multilayered microfluidic devices with a micronozzle array structure have been developed to prepare unique hydrogel microfibers with highly complex cross-sectional morphologies. Hydrogel precursor solutions with different compositions are introduced through vertical micronozzles, united and focused, and continuously gelled to form hydrogel fibers with multiple regions of different physicochemical composition. We prepared alginate hydrogel microfibers with diameters of 60 ~ 130 µm and 4/8 parallel regions in the periphery. Neuron-like PC12 cells encapsulated in the parallel region, which was made of a soft hydrogel matrix, proliferated and formed linear intercellular networks along the fiber length because of the physical restrictions imposed by the relatively rigid regions. After cultivation for 14 days, one-millimeter-long intercellular networks that structurally mimic complex nerve bundles found in vivo were formed. The proposed fibers should be useful for producing various in vivo linear tissues and should be applicable to regenerative medicine and physiological studies of cells.

Preparation of stripe-patterned heterogeneous hydrogel sheets using microfluidic devices for high-density coculture of hepatocytes and fibroblasts.

Here we demonstrate the production of stripe-patterned heterogeneous hydrogel sheets for the high-density 3D coculture of multiple cell types, by using microchannel-combined micronozzle devices. The prepared hydrogel sheet, composed of multiple regions with varying physical stiffness, regulates the direction of proliferation of encapsulated cells and enables the formation of arrays of rod-like heterotypic organoids inside the hydrogel matrix. We successfully prepared stripe-patterned hydrogel sheets with a uniform thickness of ~100 µm and a width of several millimeters. Hepatoma cells (HepG2) and fibroblasts (Swiss 3T3) were embedded inside the hydrogel matrix and cocultured, to form heterotypic micro-organoids mimicking in vivo hepatic cord structures. The upregulation of hepatic functions by the 3D coculture was confirmed by analyzing liver-specific functions. The presented heterogeneous hydrogel sheet could be useful, as it provides relatively large, but precisely-controlled, 3-dimensional microenvironments for the high-density coculture of multiple types of cells.