Non-adhesive Areas Research Articles

O-064 Can artificial removal of the zona pellucida with severe fragmentation improve culture and pregnancy outcomes?

Abstract Study question Can artificial removal of the zona pellucida (ZP-free) in patients whose embryos had severe fragmentation improve culture and pregnancy outcomes? Summary answer In patients with severe fragmentation, blastulation and live birth rate were improved by ZP-free procedure, and 14 live babieshave been obtained so far. What is known already In 2020, Yumoto et al. first reported that ZP-free procedure at the pronuclear stage decreased the rate of fragmentation and improved blastulation of 3 pronuclei embryos. Therefore, the ZP may not always be necessary for normal embryonic development after the pronuclear stage. The same group presented at the 37th ESHRE meeting in 2021, that the transfer of blastocyst derived from 2 pronuclei embryos (2PN) after ZP-free procedure resulted in normal pregnancy and live births in two patients who had had poor-quality embryos only before ZP-free procedure. We also presented culture outcomes improved by ZP-free procedure at the 2023 ESHRE meeting. Study design, size, duration This study included 571 embryos derived from 2PN on 118 cycles in 61 couples obtained between July 2021 and November 2023, whose previous oocyte retrieval cycle had shown massive cytoplasmic fragmentation at the first cleavage confirmed by a time-laps incubator (TLI) in two institutes. Mean age of women was 40.0±3.8 years. Culture and pregnancy outcomes after blastocyst transfer were compared with 1272 embryos derived from 2PN in the past 247 cycles without ZP-free procedure (ZP-intact). Participants/materials, setting, methods All participants provided written informed consent, and IRB approval was obtained. Embryos were placed in 0.125M sucrose-containing HEPES for cytoplasm shrinking at the pronuclear stage. The ZP was partially removed in non-adhesive areas with a laser system. Ooplasms were completely separated from ZP by blowing the medium to adhesion point like a jet car wash. The ZP-free embryos were cultured in TLI up to 7 days, followed by blastocyst cryopreservation for future embryo transfer cycles. Main results and the role of chance Day 3 embryos were divided into 3 groups using the Veeck’s classification: 8 cell embryos with grade 2 or higher were defined as good, embryos with grade 4 or 5 regardless of the number of cells were defined as poor, and others were defined as fair. We compared the culture outcomes of ZP-free cycle and previous ZP-intact cycle. For day 3 embryos, good, fair and poor grade were 15.6%, 55.3% and 29.1% in ZP-free embryos compared with 3.7%, 42.5%, and 53.8% in ZP-intact embryos, respectively (p < 0.001). As to the blastocysts, blastocysts development and good-quality blastocysts (G3BB or more on day 5 by the Gardner’s classification) rates were 36.9% and 19.4% in ZP-free embryos compared with 23.1% and 6.0% in ZP-intact embryos, respectively (p < 0.001). Clinical pregnancy rates for ZP-free and ZP-intact blastocyst transfer were 36.1% (22/61) and 15.0% (17/113), miscarriage rates were 18.2% (4/22) and 82.4% (14/17), and ongoing pregnancy rates were 29.5% (18/61) and 2.7% (3/113), respectively, with statistical significance after ZPF procedure (P < 0.01). Fourteen live babies have given birth by single blastocyst transfer with ZP-free procedure so far. Limitations, reasons for caution This study was retrospective study without any control group and was conducted at only two fertility centers. It is necessary to conduct sibling oocytes study or extensive randomized controlled trial to create criteria for patients who are benefit from ZP-free procedure. Moreover, safety of ZP-free culture should be evaluated. Wider implications of the findings ZP-free procedure in patients with severe fragmentated embryos at the pronuclear stage, reduced fragmentation and improved blastocyst development and clinical outcomes. We suggest that ZP-free embryos could be proposed as a new treatment option for patients who have had poor quality embryos with severe fragmentation. Trial registration number Not applicable

Secondary and tertiary transfer of latent fingermarks using a sticky note – A feasibility study

Latent fingermarks are enhanced in order to be visible and available for comparison to determine source. Once a fingermark has been identified to a source, the activity that led to it being left on a particular surface may need to be determined. It has been previously shown that under certain conditions fingermarks initially deposited onto a surface (the primary transfer) can be transferred on to another substrate through direct contact – secondary transfer. This study investigates the possibility of secondary and subsequent tertiary transfer using sticky notes. To explore secondary transfer, fingermarks were deposited directly onto two different brands of sticky notes, spanning the adhesive and non-adhesive areas, and then placed in direct contact with paper for up to 72 h under a 5 kg weight. For some donors, there was transfer of fingermarks from the sticky note to the paper, with better results for the adhesive areas. The quality of the transferred fingermarks was dependent on initial fingermark quality and the transferred fingermark was a mirror image of the original. The type of paper used as the secondary substrate was also shown to have an effect. Given the adhesive nature of sticky notes tertiary transfer was also investigated and the potential to lift fingermarks from a glass slide and transfer them onto paper or a second glass slide. In the case of transfer to paper, there were only tertiary transferred fingermarks considered to be of useful quality (score 3 or 4) in 6% of samples and a further 33% of samples were detected but provided evidence of contact only (score 1 or 2) (n = 120). For transfer to glass, tertiary transferred samples were of poorer quality with no useful fingermarks and only 3% of samples scoring 1 or 2 (n = 120). The latter was in part due to the deposition of sticky note adhesive traces obscuring the fingermarks. In the case of tertiary transfer, fingermarks on the final tertiary surface were in the correct orientation. This work demonstrates that whilst tertiary transfer of fingermarks is possible under the laboratory conditions used, the likelihood of the effective transfer of a useful and potentially identifiable fingermark is in reality low.

Cell behaviors within a confined adhesive area fabricated using novel micropatterning methods.

In the field of cell and tissue engineering, there is an increasing demand for techniques to spatially control the adhesion of cells to substrates of desired sizes and shapes. Here, we describe two novel methods for fabricating a substrate for adhesion of cells to a defined area. In the first method, the surface of the coverslip or plastic dish was coated with Lipidure, a non-adhesive coating material, and air plasma was applied through a mask with holes, to confer adhesiveness to the surface. In the second method, after the surface of the coverslip was coated with gold by sputtering and then with Lipidure; the Lipidure coat was locally removed using a novel scanning laser ablation method. These methods efficiently confined cells within the adhesive area and enabled us to follow individual cells for a longer duration, compared to the currently available commercial substrates. By following single cells within the confined area, we were able to observe several new aspects of cell behavior in terms of cell division, cell–cell collisions, and cell collision with the boundary between adhesive and non-adhesive areas.

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells.

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley's K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Extracellular Matrix Geometry and Initial Adhesive Position Determine Stress Fiber Network Organization during Cell Spreading

Three-dimensional matrices often contain highly structured adhesive tracks that require cells to turn corners and bridge non-adhesive areas. Here, we investigate these complex processes using micropatterned cell adhesive frames. Spreading kinetics on these matrices depend strongly on initial adhesive position and are predicted by a cellular Potts model (CPM), which reflects a balance between adhesion and intracellular tension. As cells spread, new stress fibers (SFs) assemble periodically and parallel to the leading edge, with spatial intervals of ∼2.5μm, temporal intervals of ∼15min, and characteristic lifetimes of ∼50min. By incorporating these rules into the CPM, we can successfully predict SF network architecture. Moreover, we observe broadly similar behavior when we culture cells on arrays of discrete collagen fibers. Our findings show that ECM geometry and initial cell position strongly determine cell spreading and that cells encode a memory of their spreading history through SF network organization.

Single Cell Microarrays Fabricated by Microscale Plasma-Initiated Protein Patterning (μPIPP).

Micropatterned arrays considerably advanced single cell fluorescence time-lapse measurements by providing standardized boundary conditions for thousands of cells in parallel. In these assays, cells are forced to adhere to defined microstructured protein islands separated by passivated, nonadhesive areas. Here we provide a detailed protocol on how to reproducibly fabricate high quality single cell arrays by microscale plasma-initiated protein patterning (μPIPP). Advantages of μPIPP arrays are the ease of preparation and the unrestricted choice of substrates as well as proteins. We demonstrate how the arrays enable the efficient measurement of single cell time trajectories using automated data acquisition and data analysis by example of single cell gene expression after mRNA transfection and time courses of single cell apoptosis. We discuss the more general use of the protocol for assessment of single cell dynamics with the help of fluorescent reporters.

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 µm diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

Directing cell migration using micropatterned and dynamically adhesive polymer brushes

Micropatterning techniques, such as photolithography and microcontact printing, provide robust tools for controlling the adhesive interactions between cells and their extracellular environment. However, the ability to modify these interactions in real time and examine dynamic cellular responses remains a significant challenge. Here we describe a novel strategy to create dynamically adhesive, micropatterned substrates, which afford precise control of cell adhesion and migration over both space and time. Specific functionalization of micropatterned poly(ethylene glycol methacrylate) (POEGMA) brushes with synthetic peptides, containing the integrin-binding arginine–glycine–aspartic acid (RGD) motif, was achieved using thiol–yne coupling reactions. RGD activation of POEGMA brushes promoted fibroblast adhesion, spreading and migration into previously non-adhesive areas, and migration speed could be tuned by adjusting the surface ligand density. We propose that this technique is a robust strategy for creating dynamically adhesive biomaterial surfaces and a useful assay for studying cell migration.

Probabilistic density maps to study the spatial organization of endocytosis.

Despite a large body of publications on endocytosis, only a few studies have focused on its spatial organization. To study how endocytosis is related to distinct cellular sites, we combine cell normalization by the "micropatterning technique" with the quantification of spatial organization by "probabilistic density mapping." Micropatterns of extracellular matrix proteins impose adhesive and non-adhesive areas to cultured cells and allow the control of adhesion geometry, shape, and cell organization. Probabilistic density maps provide a visual summary for 3D localization of the structures of interest and enable the extraction of robust statistics for quantification of cellular organization. Here, we provide a method to analyze and compare the spatial distribution of endocytosed ligands in micropatterned cells. This approach permits to establish the role of cellular adhesion on uptake mechanisms and to address the potential function of predefined sites of endocytosis.

Efficient creation of cellular micropatterns with long-term stability and their geometric effects on cell behavior

Cellular micropatterning with bio-adhesive and nonadhesive areas has attracted increasing interest for the precise design of cell-to-surface attachment in cell biology studies, tissue engineering, cell-based biosensors, biological assays, and drug development and screening. In this paper we describe a simple and efficient method to create a two-dimensional stable cellular microenvironment, which is based on (1) forming a protein-resistant oligo(ethylene glycol) methyl ether methacrylate polymer layer on the substrates via surface-initiated atom transfer radical polymerization; (2) placing a defined photomask on the substrate and exposing the substrate to ultraviolet light; and (3) immersing the patterned surface in a fibronectin solution to form cell-adhesive protein patterns in a cell-resistant background. The resulting surfaces are tailored into cell-adhesive and cell-resistant regions. Three different types of cells (NIH-3T3, PC12, bone marrow-derived mesenchymal stem cells) are seeded on such patterned surfaces to form cellular patterns. The geometric effects on cell behavior are investigated. The long-term stability is tested by NIH-3T3 fibroblasts and mesenchymal stem cells and excellent retention of cellular patterns is observed. The strategy illustrated here offers an efficient way to create a stable, patterned cellular microenvironment, and could be employed in tissue engineering to study the effect of micropatterns on the proliferation and differentiation of cells, and in particular mesenchymal stem cells.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity

Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell-cell or cell-extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus-centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes.

Cell distribution of stress fibres in response to the geometry of the adhesive environment

Cells display a large variety of shapes when plated in classical culture conditions despite their belonging to a common cell type. These shapes are transitory, since cells permanently disassemble and reassemble their cytoskeleton while moving. Adhesive micropatterns are commonly used to confine cell shape within a given geometry. In addition the micropattern can be designed so as to impose cells to spread upon adhesive and nonadhesive areas. Modulation of the pattern geometry allows the analysis of the mechanisms governing the determination of cell shape in response to external adhesive conditions. In this study, we show that the acquisition of cell shape follows two stages where initially the cell forms contact with the micropattern. Here, the most distal contacts made by the cell with the micropattern define the apices of the cell shape. Then secondly, the cell borders that link two apices move so as to minimise the distance between the two apices. In these cell borders, the absence of an underlying adhesive substrate is overcome by stress fibres forming between the apices, which in turn are marked by an accumulation of focal adhesions. By inhibiting myosin function, cell borders on nonadhesive zones become more concave, suggesting that the stress fibres work against the membrane tension in the cell border. Moreover, this suggested that traction forces are unevenly distributed in stationary, nonmigrating, cells. By comparing the stress fibres in cells with one, two, or three nonadherent cell borders it was reasoned that stress fibre strength is inversely proportional to number. We conclude that cells of a given area can generate the same total sum of tractional forces but that these tractional forces are differently spaced depending on the spatial distribution of its adherence contacts.

Patterned cell culture inside microfluidic devices

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.

A novel method to prepare size‐regulated spheroids composed of human dermal fibroblasts

A simple method to prepare size-regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly-N-isopropyl-acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37 degrees C, then when lowering the temperature of culture medium the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30 degrees C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV-irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 microg/cm(2) (collagen ratio, 4.5%), UV-irradiation energy of 2000 J/m(2) was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV-irradiation, it was possible to obtain cell-adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 microm in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 microm in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size-regulated spheroids for different applications; for example, in toxicology tests.