Exploring the influence of silicon oxide microchips shape on cellular uptake using imaging flow cytometry.

Nano- and micro-carriers of therapeutic molecules offer numerous advantages for drug delivery, and the shape of these particles plays a vital role in their biodistribution and their interaction with cells. However, analysing how microparticles are taken up by cells presents methodological challenges. Qualitative methods like microscopy provide detailed imaging but are time-consuming, whereas quantitative methods such as flow cytometry enable high-throughput analysis but struggle to differentiate between internalised and surface-bound particles. Instead, imaging flow cytometry combines the best of both worlds, offering high-resolution imaging with the efficiency of flow cytometry, allowing for quantitative analysis at the single-cell level. This study focuses on fluorescently labelled silicon oxide microchips of various morphologies but related surface areas and volumes: rectangular cuboids and apex-truncated square pyramid microchips fabricated using photolithography techniques, offering a reliable basis for comparison with the more commonly studied spherical particles. Imaging flow cytometry was utilised to evaluate the effect of particle shape on cellular uptake using RAW 264.7 cells and revealed phagocytosis of particles with all shapes. Increasing the particle dose enhanced the uptake, while macrophage stimulation had minimal effect. Using a ratio particle:cell of 10:1 cuboids and spheres showed an uptake rate of approximately 50%, in terms of the percentage of cells with internalised particles, and the average number of particles taken up per cell ranging from about 1-1.5 particle/cell for all the different shapes. This study indicates how differently shaped micro-carriers offer insights into particle uptake variations, demonstrating the potential of non-spherical micro-carriers for precise drug delivery applications.

Poorly Differentiated Scrotal Carcinoma With Apocrine Immunophenotype.

ABSTRACT: Cutaneous carcinoma of the scrotum is rare with the most common type being squamous cell carcinoma. Here, we report 6 cases of poorly differentiated carcinoma with apocrine immunophenotype. Mean age at presentation was 68 years (range: 31-91 years). Clinical presentation included eczematous rash over mass, scrotal cyst, ulcerated mass, and mass. Tumor size ranged from 1.2 to 5.5 cm (average 2.5 cm). The tumors were solid with involvement of the dermis/hypodermis and composed of cords and nests of eosinophilic cells displaying nuclei with prominent nucleoli and surrounded by desmoplastic stroma. Focal squamous differentiation was evident in one case (17%). An intraductal component was seen in one case (17%). Pagetoid spread in the epidermis was seen in 3 cases. There was no morphologic evidence of apocrine differentiation. By immunohistochemistry, the tumor cells were positive for GCDFP-15 (n = 6/6), GATA3 (n = 6/6), CK7 (n = 5/5), AR (n = 4/4), and mammaglobin (n = 3/5). Five (83%) patients had metastases at diagnosis. Treatment included wide local excisions and inguinal lymph node dissection, followed by chemotherapy (gemcitabine, carboplatin; n = 3), trastuzumab/Lupron (n = 1), tamoxifen/Arimidex (n = 1), and radiotherapy (n = 1). Two patients (40%) were dead of disease, less than 2 years from diagnosis. Four patients developed metastases to lymph nodes, liver, bones, and lungs. Molecular analysis (n = 2) detected a HER-2 mutation in one and microsatellite instability in another. Although the presence of an intraepidermal pagetoid component could hint toward the diagnosis of invasive extramammary Paget disease, tumors without an intraepidermal component could be diagnostically challenging given the lack of morphologic evidence of apocrine differentiation.

Tracking intracellular forces and mechanical property changes in mouse one-cell embryo development.

Cells comprise mechanically active matter that governs their functionality, but intracellular mechanics are difficult to study directly and are poorly understood. However, injected nanodevices open up opportunities to analyse intracellular mechanobiology. Here, we identify a programme of forces and changes to the cytoplasmic mechanical properties required for mouse embryo development from fertilization to the first cell division. Injected, fully internalized nanodevices responded to sperm decondensation and recondensation, and subsequent device behaviour suggested a model for pronuclear convergence based on a gradient of effective cytoplasmic stiffness. The nanodevices reported reduced cytoplasmic mechanical activity during chromosome alignment and indicated that cytoplasmic stiffening occurred during embryo elongation, followed by rapid cytoplasmic softening during cytokinesis (cell division). Forces greater than those inside muscle cells were detected within embryos. These results suggest that intracellular forces are part of a concerted programme that is necessary for development at the origin of a new embryonic life.

Traceability of human sperm samples by direct tagging with polysilicon microbarcodes.

The increasing number of patients undergoing assisted reproductive technology (ART) treatments and of cycles performed in fertility centres has led to some traceability errors. Although the incidence of mismatching errors is extremely low, any error is unacceptable, therefore different strategies have been developed to further minimize these errors, such as manual double-witnessing or electronic witnessing systems. More recently, our group developed a direct tagging method consisting of attaching microbarcodes directly to the zona pellucida of human oocytes/embryos. Here, this method is taken a step further by using these microbarcodes to tag human semen samples, demonstrating that the barcodes are not toxic and do not interfere in the selection of motile spermatozoa nor in the cryopreservation of the sperm samples. In addition, when this tagging system was applied to an animal model (rabbit), pregnancy rate and kitten viability were not affected.

Barcode tagging of human oocytes and embryos to prevent mix-ups in assisted reproduction technologies.

STUDY QUESTION: Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of human oocytes and embryos during assisted reproduction technologies (ARTs)? SUMMARY ANSWER: The direct tagging system based on lectin-biofunctionalized polysilicon barcodes of micrometric dimensions is simple, safe and highly efficient, allowing the identification of human oocytes and embryos during the various procedures typically conducted during an assisted reproduction cycle. WHAT IS KNOWN ALREADY: Measures to prevent mismatching errors (mix-ups) of the reproductive samples are currently in place in fertility clinics, but none of them are totally effective and several mix-up cases have been reported worldwide. Using a mouse model, our group has previously developed an effective direct embryo tagging system which does not interfere with the in vitro and in vivo development of the tagged embryos. This system has now been tested in human oocytes and embryos. STUDY DESIGN, SIZE, DURATION: Fresh immature and mature fertilization-failed oocytes (n = 21) and cryopreserved day 1 embryos produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (n = 205) were donated by patients (n = 76) undergoing ARTs. In vitro development rates, embryo quality and post-vitrification survival were compared between tagged (n = 106) and non-tagged (control) embryos (n = 99). Barcode retention and identification rates were also calculated, both for embryos and for oocytes subjected to a simulated ICSI and parthenogenetic activation. Experiments were conducted from January 2012 to January 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Barcodes were fabricated in polysilicon and biofunctionalizated with wheat germ agglutinin lectin. Embryos were tagged with 10 barcodes and cultured in vitro until the blastocyst stage, when they were either differentially stained with propidium iodide and Hoechst or vitrified using the Cryotop method. Embryo quality was also analyzed by embryo grading and time-lapse monitoring. Injected oocytes were parthenogenetically activated using ionomycin and 6-dimethylaminopurine. MAIN RESULTS AND THE ROLE OF CHANCE: Blastocyst development rates of tagged (27/58) and non-tagged embryos (24/51) were equivalent, and no significant differences in the timing of key morphokinetic parameters and the number of inner cell mass cells were detected between the two groups (tagged: 24.7 ± 2.5; non-tagged: 22.3 ± 1.9), indicating that preimplantation embryo potential and quality are not affected by the barcodes. Similarly, re-expansion rates of vitrified-warmed tagged (19/21) and non-tagged (16/19) blastocysts were similar. Global identification rates of 96.9 and 89.5% were obtained in fresh (mean barcode retention: 9.22 ± 0.13) and vitrified-warmed (mean barcode retention: 7.79 ± 0.35) tagged embryos, respectively, when simulating an automatic barcode reading process, though these rates were increased to 100% just by rotating the embryos during barcode reading. Only one of the oocytes lost one barcode during intracytoplasmic injection (100% identification rate) and all oocytes retained all the barcodes after parthenogenetic activation. LIMITATIONS, REASONS FOR CAUTION: Although the direct embryo tagging system developed is effective, it only allows the identification and traceability of oocytes destined for ICSI and embryos. Thus, the traceability of all reproductive samples (oocytes destined for IVF and sperm) is not yet ensured. WIDER IMPLICATIONS OF THE FINDINGS: The direct embryo tagging system developed here provides fertility clinics with a novel tool to reduce the risk of mix-ups in human ARTs. The system can also be useful in research studies that require the individual identification of oocytes or embryos and their individual tracking. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Sociedad Española de Fertilidad, the Spanish Ministry of Education and Science (TEC2011-29140-C03) and the Generalitat de Catalunya (2009SGR-00282 and 2009SGR-00158). The authors do not have any competing interests.

Identification of bovine embryos cultured in groups by attachment of barcodes to the zona pellucida.

The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each individual female have to be cultured individually or in very small groups. However, it has been demonstrated that single-embryo culture is less efficient than embryo culture in groups. To overcome this limitation, we developed a direct embryo-tagging system, which allows the collective culture of embryos from different origins whilst preserving their pedigree. Presumptive bovine zygotes were tagged with eight wheat-germ agglutinin biofunctionalised polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Four different barcodes were used to encode groups of 20-25 embryos, which were then cultured in the same drop. Cleavage, Day-7 and Day-8 blastocysts and barcode retention rates were assessed. In addition, Day-7 blastocysts were vitrified and warmed. Barcode attachment to the ZP of bovine embryos affected neither in vitro embryo development nor post-warming survival of the tagged embryos. All the embryos maintained barcodes attached until Day 8 of culture (3.63±0.37 barcodes per embryo) and could be identified. In conclusion, identification of embryos by barcodes attached to the ZP is feasible and will allow the culture of embryos from different donors in the same drop.

Cuboids Prevail When Unraveling the Influence of Microchip Geometry on Macrophage Interactions and Metabolic Responses.

Drug delivery advances rely on using nano- and microsized carriers to transfer therapeutic molecules, although challenges persist in increasing the availability of new and even approved pharmaceutical products. Particle shape, a critical determinant in how these carriers distribute within the body after administration, raises opportunities of using, for instance, micrometer-sized nonspherical particles for vascular targeting and thereby creating new prospects for precise drug delivery to specific targeted areas. The versatility of polycrystalline silicon microfabrication allows for significant variation in the size and shape of microchips, and so, in the current work, photolithography was employed to create differently shaped polysilicon microchips, including cuboids, cubes, bars, and cylinders, to explore the influence of particle shape on cellular interactions. These microchips with different shapes and lateral dimensions, accounting for surface areas in the range of ca. 15 to 120 µm2 and corresponding total volumes of 0.4 to 27 µm3, serve as ideal models for investigating their interactions with macrophages with diameters of ca. 20 µm. Side-scattering imaging flow cytometry was employed for studying the interaction of label-free prepared microchips with RAW 264.7 macrophages. Using a dose of 3 microchips per cell, results show that cuboids exhibit the highest cellular association (ca. 25%) and uptake (ca. 20%), suggesting their potential as efficient carriers for targeted drug delivery to macrophages. Conversely, similarly sized cylinders and bar-shaped microchips exhibit lower uptakes of about 8% and about 6%, respectively, indicating potential benefits in evading macrophage recognition. On average, 1-1.5 microchips were internalized, and ca. 1 microchip was surface-bound per cell, with cuboids showing the higher values overall. Macrophages respond to microchips by increasing their metabolic activity and releasing low levels of intracellular enzymes, indicating reduced toxicity. Interestingly, increasing the particle dose enhances macrophage metabolic activity without significantly affecting enzyme release.

Lymphomatoid Papulosis With T-cell Receptor-Gamma Delta Expression: A Clinicopathologic Case-series of 26 Patients of an Underrecognized Immunophenotypic Variant of Lymphomatoid Papulosis.

Lymphomatoid papulosis (LyP) has several histopathologic presentations. LyP featuring gamma-delta (γδ) T-cell receptor expression may masquerade as and may be misdiagnosed as aggressive cutaneous T-cell lymphoma, particularly primary cutaneous γδ T-cell lymphoma (PCGDTL) or γδ mycosis fungoides. We performed a clinicopathologic analysis of the largest series of LyP featuring γδ T-cell expression. We identified 26 patients with a diagnosis of LyP with γδ T cells from our institutions, as well as through a comprehensive review of the literature, and characterized these cases. Most cases were treated with topical steroids or not treated at all. The majority of cases showed a CD4 - CD8 + phenotype and featured at least one cytotoxic marker. Histopathologic features included an intraepidermal or dermal infiltrate with large cells and frequent angiotropism. One case was initially misdiagnosed as PCGDTL, requiring further therapy. Our case series, the largest international cohort of γδ T cell predominant LyP cases, confirms marked clinicopathologic heterogeneity that may contribute to misdiagnosis, reasserting the need to identify classic clinical features, CD30 + T-cell components, and markers of cytotoxicity when dealing with this differential diagnosis. A limitation of this study includes somewhat limited follow-up, histologic, and immunophenotypic information for some cases.

Direct embryo tagging and identification system by attachment of biofunctionalized polysilicon barcodes to the zona pellucida of mouse embryos.

STUDY QUESTION: Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos? SUMMARY ANSWER: The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies. WHAT IS KNOWN ALREADY: Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. STUDY DESIGN, SIZE, DURATION: Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n=140) and after embryo cryopreservation (n = 84). Finally, the full-term development of tagged embryos was evaluated (n =105). PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing. MAIN RESULTS AND THE ROLE OF CHANCE: Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading system, although these rates could be increased to 100% by simply rotating the embryos during the reading process. LIMITATIONS, REASONS FOR CAUTION: The direct embryo tagging developed here has exclusively been tested in mouse embryos. Its effectiveness in other species, such as the human, is currently being tested. WIDER IMPLICATIONS OF THE FINDINGS: The direct embryo tagging system developed here, once tested in human embryos, could provide fertility clinics with a novel tool to reduce the risk of mix-ups in human assisted reproduction technologies.

Hydroa vacciniforme-like cutaneous T-cell lymphoma: clinicopathologic and immunohistochemical study of 12 cases.

BACKGROUND: Hydroa vacciniforme (HV)-like lymphoma (HVL) is a rare and aggressive cutaneous T-cell lymphoma occurring mainly in children in Latin America and Asia. Chronic latent Epstein-Barr virus infection has been associated with both HV and HVL. OBJECTIVE: We sought to evaluate the clinical presentation and histopathology of this rare cutaneous T-cell lymphoma. METHODS: We reviewed the clinical, morphologic, and immunophenotypical features in 12 cases of HVL from Bolivia. RESULTS: All 12 patients had skin lesions in both sun-exposed and nonsun-exposed areas, including edema, blistering, ulceration, and scarring, with a slowly progressive relapsing course. All 12 patients presented with systemic symptoms and showed a characteristic swelling of the nose and lips, and periorbital edema. Eight patients died an average of 5.3 months after initial diagnosis. Four patients remained alive with persistent disease. Histopathologic examination showed an atypical lymphocytic infiltrate with angiotropism and angiocentricity. The immunophenotype showed a cytotoxic T-cell (CD8(+)) profile. All cases were associated with Epstein-Barr virus infection and differed clinically from other forms of cutaneous T-cell lymphoma. LIMITATIONS: Only a limited number of cases were studied. CONCLUSIONS: This study confirms that HVL is a highly aggressive lymphoma, although some patients have a more indolent, chronic course.

Atypical melanocytic lesions of the thigh with spitzoid and dysplastic features: a clinicopathologic study of 29 cases.

Melanocytic nevi in certain anatomic locations can display unusual histopathologic features potentially creating diagnostic uncertainty. Benign melanocytic nevi in sites such as acral, genital, and flexural areas may show unusual architecture and cytologic atypia, which can mimic dysplastic nevi and, sometimes, melanoma. Twenty-nine benign melanocytic skin lesions were identified in the thigh of 26 women and 3 men who showed atypical histologic features, including both dysplastic and spitzoid features. All lesions measured less than 1 cm in diameter. Eighteen cases showed features of compound nevi, and 11 were junctional. In all cases, the lesions displayed spitzoid features including large epithelioid and/or spindle cells, some melanocytes with ganglion-like cytomorphology, and focal suprabasilar upward migration. All cases showed dysplastic features. Clinical follow-up was available in 26 patients; no recurrences or metastases were observed. The demographic features and the anatomical sites of these melanocytic nevi appear to be reproducible, and in the assessment of histologically difficult cases, these data are helpful. It is our opinion that the malignant potential of these lesions with hybrid patterns (spitzoid/dysplastic) has yet to be determined, and although we favor these lesions to be benign, longer follow-up may yield a more complete understanding of their biologic potential.

Prevention of incisional hernia after kidney transplantation: study protocol for a randomized controlled trial.

BACKGROUND: Incisional hernia is a common complication after kidney transplantation with an incidence of 1.6-18%. Concerning non-transplant patients, a recently published meta-analysis describes a reduction of the incidence of incisional hernia of up to 85% due to prophylactic mesh replacement in elective, midline laparotomy. The aim of our study is to show a reduction of the incidence of incisional hernia after kidney transplantation with minimal risk for complication. METHODS/DESIGN: This is a blinded, randomized controlled trial comparing time to incisional hernia over a period of 24 months between patients undergoing kidney transplantation and standardized abdominal closure with or without prophylactic placement of ProGrip™ (Medtronic, Fridley, MN, USA) mesh in an onlay position. As we believe that the mesh intervention is superior to the standard procedure in reducing the incidence of hernia, this is a superiority trial. DISCUSSION: The high risk for developing incisional hernia following kidney transplantation might be reduced by prophylactic mesh placement. ProGrip™ mesh features polylactic acid (PLA) microgrips that provide immediate, strong and uniform fixation. The use of this mesh combines the effectiveness demonstrated by the macropore propylene meshes in the treatment of incisional hernias, a high simplicity of use provided by its capacity for self-fixation that does not increase significantly surgery time, and safety. TRIAL REGISTRATION: ClinicalTrials.gov NCT04794582. Registered on 08 March 2021. Protocol version 2.0. (02-18-2021).

Efficient biofunctionalization of polysilicon barcodes for adhesion to the zona pellucida of mouse embryos.

Cell tracking is an emergent area in nanobiotechnology, promising the study of individual cells or the identification of populations of cultured cells. In our approach, microtools designed for extracellular tagging are prepared, because using biofunctionalized polysilicon barcodes to tag cell membranes externally avoids the inconveniences of cell internalization. The crucial covalent biofunctionalization process determining the ultimate functionality was studied in order to find the optimum conditions to link a biomolecule to a polysilicon barcode surface using a self-assembled monolayer (SAM) as the connector. Specifically, a lectin (wheat germ agglutinin, WGA) was used because of its capacity to recognize some specific carbohydrates present on the surface of most mammalian cells. Self-assembled monolayers were prepared on polysilicon surfaces including aldehyde groups as terminal functions to study the suitability of their covalent chemical bonding to WGA. Some parameters, such as the polysilicon surface roughness or the concentration of WGA, proved to be crucial for successful biofunctionalization and bioactivity. The SAMs were characterized by contact angle measurements, time-of-flight secondary ion mass spectrometry (TOF-SIMS), laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS), and atomic force microscopy (AFM). The biofunctionalization step was also characterized by fluorescence microscopy and, in the case of barcodes, by adhesion experiments to the zona pellucida of mouse embryos. These experiments showed high barcode retention rates after 96 h of culture as well as high embryo viability to the blastocyst stage, indicating the robustness of the biofunctionalization and, therefore, the potential of these new microtools to be used for cell tagging.

How has the COVID-19 pandemic influenced prostate cancer?-a tertiary single-centre analysis of oncological results, diagnosis and treatment times.

Background: The coronavirus disease 2019 (COVID-19) pandemic has affected care for diseases like cancer. The aim was to evaluate the impact of COVID-19 on waiting times for diagnosis and treatment of prostate cancer (PC), as well as the possible effect on the treatment results in PC patients undergoing radical prostatectomy. Methods: We compared the results of 497 patients who underwent biopsy prior to the COVID-19 pandemic (1 January-31 December 2019) with those of 290 patients biopsied during the COVID-19 pandemic (1 January-31 December 2020). Demographic data, tumour characteristics, type of treatment and diagnosis times were comparable. Prostate specific antigen (PSA) levels were recorded at consultation prior to biopsy and after treatment. Mann-Whitney and chi-square tests were used to compare continuous variables and percentages, respectively. Results: In 2020, there were fewer urology consultations (35,160 vs. 40,225 in 2019). The median PSA in 2020 was significantly higher (14.3 vs. 9.9 ng/dL in 2019). In 2019, 53.1% (N=264) of the biopsies were positive for cancer vs. 47.2% (N=137) in 2020 (P=0.104). In 2020, more patients presented with metastatic disease (7.3% vs. 1.9%, P=0.009). Also, in 2020 there was a longer waiting time for prostate biopsy (42.1 vs. 35.3 days in 2019, P=0.019). A total of 132 patients underwent laparoscopic radical prostatectomy (LARP). The median time until surgery was similar in both years (71.9 vs. 58.29 days). During 2020, a higher percentage of patients had ISUP grade 4 in the surgical specimen (34.3% vs. 17.5%, P=0.07). Furthermore, a higher percentage of aggressive (pT3) tumours were diagnosed (37.2% vs. 27.2%, P=0.08), and the percentage of patients with involvement of surgical margins was also higher (48.6% vs. 29.3%, P=0.027). There were no differences between the groups in terms of biochemical recurrence or persistent PSA at one year (P=0.711). Conclusions: Delayed biopsy during the COVID-19 period did not appear to adversely impact biopsy results. Patients biopsied in 2020 had higher PSA, possibly due to proper triaging. A higher rate of adverse pathology outcomes was observed in patients undergoing radical prostatectomy during the pandemic, probably due to understaging of the biopsy. This study serves to raise awareness of the risk of deterioration of care of PC patients due to possible underdiagnosis.

A novel embryo identification system by direct tagging of mouse embryos using silicon-based barcodes.

BACKGROUND: Measures to prevent assisted reproductive technologies (ART) mix-ups, such as labeling of all labware and double-witnessing protocols, are currently in place in fertility clinics worldwide. Technological solutions for electronic witnessing are also being developed. However, none of these solutions eliminate the risk of identification errors, because gametes and embryos must be transferred between containers several times during an ART cycle. Thus, the objective of this study was to provide a proof of concept for a direct embryo labeling system using silicon-based barcodes. METHODS: Three different types of silicon-based barcodes (A, B and C) were designed and manufactured, and microinjected into the perivitelline space of mouse pronuclear embryos (one to four barcodes per embryo). Embryos were cultured in vitro until the blastocyst stage, and rates of embryo development, retention of the barcodes in the perivitelline space and embryo identification were assessed every 24 h. Release of the barcodes after embryo hatching was also determined. Finally, embryos microinjected with barcodes were frozen and thawed at the 2-cell stage to test the validity of the system after cryopreservation. RESULTS: Barcodes present in the perivitelline space, independently of their type and number, did not affect embryo development rates. The majority of embryos (>90%) retained at least one of the microinjected barcodes in their perivitelline space up to the blastocyst stage. Increasing the number of barcodes per embryo resulted in a significant increase in embryo identification rates, but a significant decrease in the barcode release rates after embryo hatching. The highest rates of successful embryo identification (97%) were achieved with the microinjection of four type C barcodes, and were not affected by cryopreservation. CONCLUSIONS: Our results demonstrate the feasibility of a direct embryo labeling system and constitute the starting point in the development of such systems.

Integrating magnetic capabilities to intracellular chips for cell trapping.

Current microtechnologies have shown plenty of room inside a living cell for silicon chips. Microchips as barcodes, biochemical sensors, mechanical sensors and even electrical devices have been internalized into living cells without interfering their cell viability. However, these technologies lack from the ability to trap and preconcentrate cells in a specific region, which are prerequisites for cell separation, purification and posterior studies with enhanced sensitivity. Magnetic manipulation of microobjects, which allows a non-contacting method, has become an attractive and promising technique at small scales. Here, we show intracellular Ni-based chips with magnetic capabilities to allow cell enrichment. As a proof of concept of the potential to integrate multiple functionalities on a single device of this technique, we combine coding and magnetic manipulation capabilities in a single device. Devices were found to be internalized by HeLa cells without interfering in their viability. We demonstrated the tagging of a subpopulation of cells and their subsequent magnetic trapping with internalized barcodes subjected to a force up to 2.57 pN (for magnet-cells distance of 4.9 mm). The work opens the venue for future intracellular chips that integrate multiple functionalities with the magnetic manipulation of cells.

Focus ion beam micromachined glass pipettes for cell microinjection.

Cell handling is currently hindered by rudimentary-manufactured manipulators. Restrictive designs of glass pipettes and other micromanipulators limit functionality and often damage cells, ultimately resulting in lysis. We present a novel technique to design and mill conventional glass pipettes at specifically chosen angles and geometries. Focus ion beam milling by Ga+ ions yields extremely polished edges. Results from mouse embryo piercing correlate increased penetration rates with decreased pipette angle. Milled pipettes maintain structural integrity after repeated piercing. For the first time, the effects of unintentionally implanted Ga+ on embryo development are addressed. Optimum embryo development up to blastocyst stage after manipulation reveal little impact of residual implanted Ga+, suggesting biocompatibility and paving the way to introducing ion milling techniques in the biomedical device arena. The milling technique can be adequately tailored to specific applications and allows for mass production, presenting a promising avenue for future, increasingly demanding, cell handling.

Sharp high-aspect-ratio AFM tips fabricated by a combination of deep reactive ion etching and focused ion beam techniques.

The shape and dimensions of an atomic force microscope tip are crucial factors to obtain high resolution images at the nanoscale. When measuring samples with narrow trenches, inclined sidewalls near 90 degrees or nanoscaled structures, standard silicon atomic force microscopy (AFM) tips do not provide satisfactory results. We have combined deep reactive ion etching (DRIE) and focused ion beam (FIB) lithography techniques in order to produce probes with sharp rocket-shaped silicon AFM tips for high resolution imaging. The cantilevers were shaped and the bulk micromachining was performed using the same DRIE equipment. To improve the tip aspect ratio we used FIB nanolithography technique. The tips were tested on narrow silicon trenches and over biological samples showing a better resolution when compared with standard AFM tips, which enables nanocharacterization and nanometrology of high-aspect-ratio structures and nanoscaled biological elements to be completed, and provides an alternative to commercial high aspect ratio AFM tips.

A self-calibrating and multiplexed electrochemical lab-on-a-chip for cell culture analysis and high-resolution imaging.

In vitro analysis requires cell proliferation in conditions close to physiological ones. Lab-on-a-chip (LoC) devices simplify, miniaturize and automate traditional protocols, with the advantages of being less expensive and faster due to their shorter diffusion distances. The main limitation of current LoCs is still the control of the culture conditions. Most LoCs employ off-chip equipment to determine cell culture activity, which confers limited monitoring capacity. The few systems integrating transducers on-chip present important functional problems mostly associated with the attachment of biomolecules to the transducer surface (i.e., biofouling) and the impossibility of re-calibrating the sensors during cell culturing. This limitation is addressed in the present LoC containing a network of micro-channels and micro-chambers, which allows (i) cell seeding and cultivation, avoiding biofouling risk, (ii) multiplexed analysis of cell culture, reactivation and recalibration of the (bio)sensors without compromising cell viability, (iii) cell imaging and (iv) reference electrode compartmentalization to guarantee stability. The activity of the culture is monitored with four independent electrochemical micro-electrodes for glucose, hydrogen peroxide, conductivity and oxidation reduction potential. Electrochemical analysis is complemented with high-resolution confocal microscopy analysis. This paper demonstrates the suitability of the current configuration for cell culture monitoring and future applications in drug screening or organ-on-a-chip development.

Intracellular polysilicon barcodes for cell tracking.

During the past decade, diverse types of barcode have been designed in order to track living cells in vivo or in vitro, but none of them offer the possibility to follow an individual cell up to ten or more days. Using silicon microtechnologies a barcode sufficiently small to be introduced into a cell, yet visible and readily identifiable under an optical microscope, is designed. Cultured human macrophages are able to engulf the barcodes due to their phagocytic ability and their viability is not affected. The utility of the barcodes for cell tracking is demonstrated by following individual cells for up to ten days in culture and recording their locomotion. Interestingly, silicon microtechnology allows the mass production of reproducible codes at low cost with small features (bits) in the micrometer range that are additionally biocompatible.