Biology subjects

Hasenauer, A.

Publications and source records attributed to Hasenauer, A..

8 recordsLinked to original sources

bioRxiv2026

Extracellular matrix context shapes morphogenesis and lactation-associated states in human milk-derived mammary organoids

The mammary gland relies on reciprocal interactions between epithelial cells and their surrounding extracellular matrix (ECM) to form and maintain milk-producing tissue structures. Yet these processes remain difficult to study in human model systems. Mammary epithelial cells (MECs) can be isolated noninvasively from breast milk, but whether they generate three-dimensional organoids and respond to matrix cues has been unclear. Here, human milk-derived MECs (milk MECs) spontaneously form complex organoids, including polarized acinar and terminal duct lobular unit-like structures after isolation. To investigate how matrix composition shapes these organoids, milk MECs were cultured in decellularized mammary ECM (dECM), Matrigel, and collagen I. In dECM, milk MECs formed polarized branched networks with aligned actin organization along collagen fibrils, whereas in Matrigel they adopted a more lactation-associated state, marked by {beta}-casein expression and milk fat globules. Together, these findings establish breast milk-derived MEC organoids as a human model to study how ECM context regulates mammary morphogenesis and lactation biology.

cell biology↗

bioRxiv2026

In-Chip Volumetric Printing of Collagen-I Scaffolds for Perfusable and Stretchable Mammary Tissue Models

Engineered epithelial models require three-dimensional extracellular matrix environments that support organized cell growth and allow independent access to luminal and basal compartments. However, many organ-on-chip (OoC) fabrication strategies rely on planar geometries, non-native materials, or multi-step assembly workflows that limit architectural complexity and experimental control. Here, we report a direct in-chip volumetric printing strategy for fabricating stretchable and perfusable collagen-I scaffolds inside custom OoC devices. A vitamin C-regulated ruthenium/sodium persulfate photocrosslinking system enabled high-fidelity printing of collagen-I into open-lumen architectures with ductal- and alveolar-inspired features. By generating scaffolds directly within the final culture device, this workflow eliminates post-print transfer and integrates defined collagen architectures with compartmentalized fluidic access and a mechanically actuable chip format. To support chip-based culture, printed collagen constructs were stabilized after fabrication using EDC/NHS chemistry, which limited thermally induced collagen densification, improved shape retention, and maintained scaffold anchorage during perfusion. The chip design provided separate access to the printed lumen and surrounding basal compartment, which enabled compartment-specific fluid handling while preserving scaffold integrity during inflation, stretching, and perfusion of the printed construct. On the collagen-I scaffolds, human milk-derived mammary epithelial cells formed epithelial layers with tight junctions and lactation associated markers. The platform further supported perfusion culture, in situ staining, and whole-chip volumetric imaging. Together, this work establishes direct in-chip collagen-I volumetric printing as a biofabrication strategy for creating perfusable epithelial tissue chips with native matrix architecture and compartmentalized fluidic control.

bioengineering↗

bioRxiv2026

Vitamin C-Induced Photo-Redox Threshold Enables High-Fidelity Volumetric Printing of Pristine Collagen

Tomographic volumetric printing (TVP) enables rapid fabrication of complex, centimeter-scale 3D architectures. TVP of pristine proteins like collagen is attractive because it better preserves native bioactive motifs that regulate cell-matrix signaling. However, direct TVP of collagen remains challenging because dityrosine crosslinking, driven by visible-light-activated Ru(II)bpy32+/sodium persulfate (SPS), lacks an effective inhibitory mechanism. This results in near-immediate crosslinking upon exposure to light, which leads to an insufficient nonlinear threshold response that fails to suppress background curing. Here, we introduce vitamin C (L-ascorbic acid) as a biocompatible redox regulator to overcome this limitation. UV-Vis kinetics demonstrate that vitamin C suppresses Ru(III) accumulation and scavenges persulfate radicals within Ru/SPS system. This dual action generates a critical photo-redox and crosslinking threshold that inhibits dityrosine formation until vitamin C is depleted. Thereby the threshold response needed for TVP is successfully established, which enables high-fidelity volumetric printing of native collagen. Post-printing construct densification ([~]53% shrinkage) further improves feature resolution (80 {micro}m positive; 120 {micro}m negative) and yields mechanically stable and highly stretchable hydrogels (up to 180% strain). Collagen resin with vitamin C supports both cell seeding post-printing and cell-laden printing with high cell density and viability, enabling the rapid biofabrication of cell-instructive 3D microenvironments. Table of Contents (ToC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/717972v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@742a83org.highwire.dtl.DTLVardef@930c06org.highwire.dtl.DTLVardef@1fa7f08org.highwire.dtl.DTLVardef@aa22bb_HPS_FORMAT_FIGEXP M_FIG C_FIG Tomographic volumetric printing (TVP) of native proteins is limited by uncontrolled background crosslinking. Here, vitamin C is introduced as a biocompatible redox-regulator to establish a tunable nonlinear polymerization threshold response for TVP. This strategy effectively suppresses background crosslinking and enables high-fidelity printing of pristine collagen. Subsequent post-print densification yields robust, elastic, and cell-compatible constructs with enhanced resolution for tissue engineering applications.

bioengineering↗

bioRxiv2026

Complementary multiphoton tools to create 3D architectures in soft hydrogels for epithelial tissue engineering.

Curvature provides essential mechanical cues for epithelial cells, playing a key role in cell differentiation and morphology. Repeatable manufacture of precisely controlled curvature in soft hydrogel materials is therefore essential to study epithelial mechanobiology and function. Multiphoton (MP) based biofabrication holds promise due to its high resolution and three-dimensional design flexibility. Here, we leverage MPs advantages while increasing print speed to develop two complementary tools based on replica molding and multiphoton ablation. These can provide scalable hydrogel curvatures with tunable surface properties relevant for epithelial tissue engineering. In replica molding, MP prints are transferred into PDMS used to pattern centimeter scale arrays in hydrogels. In multiphoton ablation, hydrogels are locally degraded to generate precisely controlled curvatures and surface topography. With both methods, we repeatably guide epithelial cells into alveolar and duct-like shapes. Concave alveolar-like surfaces are shown to enhance the formation of thicker epithelial layers. We observe that surface properties, controlled by both tools, could enhance cytoskeletal organization. Using these biofabrication techniques, individual effects of curvature, surface properties, hydrogel composition, and bulk stiffness on epithelial cells can be studied. Both approaches offer high curvature control and throughput, providing a viable alternative to traditional 3D culture and other printing methods.

bioengineering↗

bioRxiv2026

A 3D printed model of human lactation

Engineering physiologically relevant breast in vitro models remains challenging due to the glands complex three-dimensional microanatomy, together with the need for epithelial polarity and hormone responsiveness. To overcome these challenges, fabrication methods are needed that rapidly create alveoli-scale structures with efficient diffusion and sustained hormonal stimulation. Here, Filamented Light (FLight) biofabrication is leveraged to print highly porous, ECM-based hydrogel scaffolds directly within standard Transwell inserts with separate apical and basal access. FLights speckle-patterned laser generates multiscale scaffold architectures that integrate filament-derived microchannels ([~]15 m) to promote diffusion with alveoli-inspired cylindrical microwell arrays (O100, O150, O200 m) that impose geometric constraints to guide epithelial organization. Each insert is printed in <10 s and incorporates slow-release prolactin microcrystals to provide lactogenic stimulation in situ. Primary human milk-derived mammary epithelial cells (milk MECs) were seeded onto the constructs. There, milk MECs line the printed microwells, establish zona occludens-1-positive tight junctions, and express lactation-associated markers (prolactin receptor and {beta}-casein), alongside milk fat globules and intracellular lipid droplets. Collectively, this rapidly reconfigurable FLight platform enables high-throughput generation of hormone-responsive human mammary microtissues for lactation-focused studies and is adaptable to other lumen-forming epithelia.

bioengineering↗

bioRxiv2025

Volumetric printed biomimetic scaffolds support in vitro lactation of human milk-derived mammary epithelial cells

The human breast is remarkably plastic and remodels with each birth to produce milk optimally suited for the changing demands of the newborn. This dynamic nature of lactation makes it challenging to study under controlled conditions. Given the health benefits of human milk, models of secretory mammary tissue would offer new opportunities to study factors that influence this important food source. First, 3D models of the mammary duct/alveoli (D/A) were designed based on shapes found in vivo. Photoresins based on mammary decellularized extracellular matrix (dECM) were optimized to match the mechanical properties of native breast tissue. Next, these dECM-based D/A models were printed with a volumetric printer and seeded with human milk-derived mammary epithelial cells (MECs). MECs formed stable epithelial layers on the printed surfaces and secreted the beta-casein and milk-fat-globules. This novel model offers exciting avenues to explore hormonal, nutritional, and mechanobiological factors involved in lactation, thereby improving understanding of lactation for the benefit of infant and their mothers.

bioengineering↗

bioRxiv2022

Synergizing algorithmic design, photoclick chemistry and multi-material volumetric printing for accelerating complex shape engineering

Accelerating the designing and manufacturing of complex shapes has been a driving factor of modern industrialization. This has led to numerous advances in computational design and modeling and novel additive manufacturing (AM) techniques that can create complex shapes for bespoke applications. By combining a new coding-based design approach with high-throughput volumetric printing, we envision a new approach to transform the way we design and fabricate complex shapes. Here, we demonstrate an algorithmic voxel-based approach, which can rapidly generate and analyze porous structures, auxetic meshes and cylinders, or perfusable constructs. We use this design scheme in conjunction with new approaches for multi-material volumetric printing based on thiol-ene photoclick chemistry to rapidly fabricate complex heterogeneous structures. Collectively, the new design and fabrication technique we demonstrate can be used across a wide-spectrum of products such as actuators, biomedical implants and grafts, or tissue and disease models. TeaserA new scheme of rapidly designing and printing complex multi-material structures for implant and tissue graft applications.

bioengineering↗

bioRxiv2022

Multiscale Hybrid Fabrication: Volumetric Printing Meets Two-Photon Ablation

The vascular tree spans length scales from centimeter to micrometer. Engineering multiscale vasculature, in particular from millimeter vessels to micrometer-size capillaries, represents an unmet challenge and may require the convergence of two or more printing modalities. Leveraging the great advances in light-based biofabrication, we herein introduce a hybrid strategy to tackle this challenge. By combining volumetric printing (VP) and high-resolution two-photon ablation (2PA), we demonstrate the possibility to create complex multiscale organotypic perfusable models with features ranging from mesoscale (VP) to microscale (2PA). To successfully combine these two methods, we first eliminated micrometer-size defects generated during VP process. Due to optical modulation instability of the laser source and selffocusing phenomenon that occurs when the light triggers the photoresin crosslinking, VP printed constructs feature micrometer-size filaments and channels. By optical tuning the refractive index of the photoresin, we demonstrate defect-free VP that can then be combined with 2PA. To facilitate the 2PA process and meet VP requirements, we introduce a purely protein-based photoclick photoresin combining gelatin-norbornene and gelatin-thiol. By optimizing defect-free VP and 2PA processes, we finally demonstrate the possibility to generate complex 3D vasculature-like constructs with features ranging from ~400 m of VP to ~2 m of 2PA. This hybrid strategy opens new possibilities to better recapitulate microtissues vasculature and complex architectures, with particular potential for microfluidics and organ/tissue-on-a-chip technologies.

bioengineering↗