Whole Tissue Imaging of Cellular Boundaries at Sub-Micron Resolutions for Automatic Cell Segmentation: Applications in Epithelial Bending of Ectodermal Appendages.

For decades, biologists have relied on confocal microscopy to understand cellular morphology and the fine details of tissue structure. However, traditional confocal microscopy of tissues have limited penetration depths of light ∼ 100 µm due to tissue opaqueness. Researchers have, thus, developed tissue clearing protocols to be used with confocal microscopy, however, current clearing protocols are not compatible with labels of cell boundaries, especially at high enough resolution to precisely segment individual cells. In this work, we devise a method to retain markers of cell boundaries, and refractive index-match the tissues with water to enable tissue imaging at high magnification using long working distance water dipping objectives. The sub-micron resolution of these images allows us to automatically segment each individual cell using a trained neural network segmentation model. These segmented images can then be utilized to quantify cell properties and morphology of the entire three-dimensional tissue. As an example application, we first test our methodology on mandibles of mutant mice that express fluorescent proteins in their membranes. We then examine a non-model animal, the catshark, and explore the cellular properties of their dental lamina and dermal denticles, which are invaginating and evaginating ectodermal structures, respectively. We, thus, demonstrate that the technique presented here provides a powerful tool to quantify, in high-throughput, the 3D structures of cells and tissues during organ morphogenesis.

Real-world persistence of multiple sclerosis disease-modifying therapies.

BACKGROUND AND PURPOSE: Treatment persistence is the continuation of therapy over time. It reflects a combination of treatment efficacy and tolerability. We aimed to describe real-world rates of persistence on disease-modifying therapies (DMTs) for people with multiple sclerosis (pwMS) and reasons for DMT discontinuation. METHODS: Treatment data on 4366 consecutive people with relapse-onset multiple sclerosis (MS) were pooled from 13 UK specialist centres during 2021. Inclusion criteria were exposure to at least one MS DMT and a complete history of DMT prescribing. PwMS in blinded clinical trials were excluded. Data collected included sex, age at MS onset, age at DMT initiation, DMT treatment dates, and reasons for stopping or switching DMT. For pwMS who had received immune reconstituting therapies (cladribine/alemtuzumab), discontinuation date was defined as starting an alternative DMT. Kaplan-Meier survival analyses were used to express DMT persistence. RESULTS: In 6997 treatment events (1.6 per person with MS), median time spent on any single maintenance DMT was 4.3 years (95% confidence interval = 4.1-4.5 years). The commonest overall reasons for DMT discontinuation were adverse events (35.0%) and lack of efficacy (30.3%). After 10 years, 20% of people treated with alemtuzumab had received another subsequent DMT, compared to 82% of people treated with interferon or glatiramer acetate. CONCLUSIONS: Immune reconstituting DMTs may have the highest potential to offer a single treatment for relapsing MS. Comparative data on DMT persistence and reasons for discontinuation are valuable to inform treatment decisions and in personalizing treatment in MS.

Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming.

We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

Engineering multicomponent tissue by spontaneous adhesion of myogenic and adipogenic microtissues cultured with customized scaffolds.

The integration of intramuscular fat-or marbling-into cultured meat will be critical for meat texture, mouthfeel, flavor, and thus consumer appeal. However, culturing muscle tissue with marbling is challenging since myocytes and adipocytes have different media and scaffold requirements for optimal growth and differentiation. Here, we present an approach to engineer multicomponent tissue using myogenic and adipogenic microtissues. The key innovation in our approach is the engineering of myogenic and adipogenic microtissues using scaffolds with customized physical properties; we use these microtissues as building blocks that spontaneously adhere to produce multicomponent tissue, or marbled cultured meat. Myocytes are grown and differentiated on gelatin nanofiber scaffolds with aligned topology that mimic the aligned structure of skeletal muscle and promotes the formation of myotubes in both primary rabbit skeletal muscle and murine C2C12 cells. Pre-adipocytes are cultured and differentiated on edible gelatin microbead scaffolds, which are customized to have a physiologically-relevant stiffness, and promote lipid accumulation in both primary rabbit and murine 3T3-L1 pre-adipocytes. After harvesting and stacking the individual myogenic and adipogenic microtissues, we find that the resultant multicomponent tissues adhere into intact structures within 6-12 h in culture. The resultant multicomponent 3D tissue constructs show behavior of a solid material with a Young's modulus of âˆ¼ 2 ± 0.4 kPa and an ultimate tensile strength of âˆ¼ 23 ± 7 kPa without the use of additional crosslinkers. Using this approach, we generate marbled cultured meat with âˆ¼ mm to âˆ¼ cm thickness, which has a protein content of âˆ¼ 4 ± 2 g/100 g that is comparable to a conventionally produced Wagyu steak with a protein content of âˆ¼ 9 ± 4 g/100 g. We show the translatability of this layer-by-layer assembly approach for microtissues across primary rabbit cells, murine cell lines, as well as for gelatin and plant-based scaffolds, which demonstrates a strategy to generate edible marbled meats derived from different species and scaffold materials.

Cell-Taxi: Mesenchymal Cells Carry and Transport Clusters of Cancer Cells.

Cell clusters that collectively migrate from primary tumors appear to be far more potent in forming distant metastases than single cancer cells. A better understanding of the collective cell migration phenomenon and the involvement of various cell types during this process is needed. Here, an in vitro platform based on inverted-pyramidal microwells to follow and quantify the collective migration of hundreds of tumor cell clusters at once is developed. These results indicate that mesenchymal stromal cells (MSCs) or cancer-associated fibroblasts (CAFs) in the heterotypic tumor cell clusters may facilitate metastatic dissemination by transporting low-motile cancer cells in a Rac-dependent manner and that extracellular vesicles secreted by mesenchymal cells only play a minor role in this process. Furthermore, in vivo studies show that cancer cell spheroids containing MSCs or CAFs have faster spreading rates. These findings highlight the active role of co-traveling stromal cells in the collective migration of tumor cell clusters and may help in developing better-targeted therapies.

Emulsion-templated microparticles with tunable stiffness and topology: Applications as edible microcarriers for cultured meat.

Cultured meat has potential to diversify methods for protein production, but innovations in production efficiency will be required to make cultured meat a feasible protein alternative. Microcarriers provide a strategy to culture sufficient volumes of adherent cells in a bioreactor that are required for meat products. However, cell culture on inedible microcarriers involves extra downstream processing to dissociate cells prior to consumption. Here, we present edible microcarriers that can support the expansion and differentiation of myogenic cells in a single bioreactor system. To fabricate edible microcarriers with a scalable process, we used water-in-oil emulsions as templates for gelatin microparticles. We also developed a novel embossing technique to imprint edible microcarriers with grooved topology in order to test if microcarriers with striated surface texture can promote myoblast proliferation and differentiation in suspension culture. In this proof-of-concept demonstration, we showed that edible microcarriers with both smooth and grooved surface topologies supported the proliferation and differentiation of mouse myogenic C2C12 cells in a suspension culture. The grooved edible microcarriers showed a modest increase in the proliferation and alignment of myogenic cells compared to cells cultured on smooth, spherical microcarriers. During the expansion phase, we also observed the formation of cell-microcarrier aggregates or 'microtissues' for cells cultured on both smooth and grooved microcarriers. Myogenic microtissues cultured with smooth and grooved microcarriers showed similar characteristics in terms of myotube length, myotube volume fraction, and expression of myogenic markers. To establish feasibility of edible microcarriers for cultured meat, we showed that edible microcarriers supported the production of myogenic microtissue from C2C12 or bovine satellite muscle cells, which we harvested by centrifugation into a cookable meat patty that maintained its shape and exhibited browning during cooking. These findings demonstrate the potential of edible microcarriers for the scalable production of cultured meat in a single bioreactor.

Photodegradable Polyacrylamide Gels for Dynamic Control of Cell Functions.

Cross-linked polyacrylamide hydrogels are commonly used in biotechnology and cell culture applications due to advantageous properties, such as the precise control of material stiffness and the attachment of cell adhesive ligands. However, the chemical and physical properties of polyacrylamide gels cannot be altered once fabricated. Here, we develop a photodegradable polyacrylamide gel system that allows for a dynamic control of polyacrylamide gel stiffness with exposure to light. Photodegradable polyacrylamide hydrogel networks are produced by copolymerizing acrylamide and a photocleavable ortho-nitrobenzyl (o-NB) bis-acrylate cross-linker. When the hydrogels are exposed to light, the o-NB cross-links cleave and the stiffness of the photodegradable polyacrylamide gels decreases. Further examination of the effect of dynamic stiffness changes on cell behavior reveals that in situ softening of the culture substrate leads to changes in cell behavior that are not observed when cells are cultured on presoftened gels, indicating that both dynamic and static mechanical environments influence cell fate. Notably, we observe significant changes in nuclear localization of YAP and cytoskeletal organization after in situ softening; these changes further depend on the type and concentration of cell adhesive proteins attached to the gel surface. By incorporating the simplicity and well-established protocols of standard polyacrylamide gel fabrication with the dynamic control of photodegradable systems, we can enhance the capability of polyacrylamide gels, thereby enabling cell biologists and engineers to study more complex cellular behaviors that were previously inaccessible using regular polyacrylamide gels.

Direct Gradient Photolithography of Photodegradable Hydrogels with Patterned Stiffness Control with Submicrometer Resolution.

Cell response to matrix mechanics is well-known; however, the ability to spatially pattern matrix stiffness to a high degree of control has been difficult to attain. This study describes the use of maskless photolithography as a flexible process for direct, noncontact gradient patterning of photodegradable hydrogels with custom graphics. Any input gray scale image can be used to directly chart hydrogel cross-link density as a function of spatial position. Hydrogels can be patterned with submicron resolution, with length scales within a single substrate spanning several orders of magnitude. A quantitative relationship between input grayscale image pixel intensity and output gel stiffness is validated, allowing for direct gradient patterning. Such physical gradient hydrogel constructs are rapidly produced in a highly controlled fashion with measured stiffness ranges and length scales that are physiologically relevant. Mesenchymal stem cells cultured on these physical gradients matrices congregate and align orthogonal to the gradient direction along iso-degraded lines. This approach results in a robust and high-throughput platform to answer key questions about cell response in heterogeneous physical environments.

Can OCT be sensitive to nanoscale structural alterations in biological tissue?

Exploration of nanoscale tissue structures is crucial in understanding biological processes. Although novel optical microscopy methods have been developed to probe cellular features beyond the diffraction limit, nanometer-scale quantification remains still inaccessible for in situ tissue. Here we demonstrate that, without actually resolving specific geometrical feature, OCT can be sensitive to tissue structural properties at the nanometer length scale. The statistical mass-density distribution in tissue is quantified by its autocorrelation function modeled by the Whittle-Matern functional family. By measuring the wavelength-dependent backscattering coefficient µb(λ) and the scattering coefficient µs, we introduce a technique called inverse spectroscopic OCT (ISOCT) to quantify the mass-density correlation function. We find that the length scale of sensitivity of ISOCT ranges from ~30 to ~450 nm. Although these sub-diffractional length scales are below the spatial resolution of OCT and therefore not resolvable, they are nonetheless detectable. The sub-diffractional sensitivity is validated by 1) numerical simulations; 2) tissue phantom studies; and 3) ex vivo colon tissue measurements cross-validated by scanning electron microscopy (SEM). Finally, the 3D imaging capability of ISOCT is demonstrated with ex vivo rat buccal and human colon samples.