Cell Culture Substrates Research Articles

Photothermal nanofiber-mediated photoporation for gentle and efficient intracellular delivery of macromolecules.

Photoporation with free photothermal nanoparticles (NPs) is a promising technology for gentle delivery of functional biomacromolecules into living cells, offering great flexibility in terms of cell types and payload molecules. However, the translational use of photoporation, such as for transfecting patient-derived cells for cell therapies, is hampered by safety and regulatory concerns as it relies on direct contact between cells and photothermal NPs. A solution is to embed the photothermal NPs in electrospun nanofibers, which form a substrate for cell culture. Here we present a protocol for photothermal electrospun nanofiber (PEN)-mediated photoporation that induces membrane permeabilization by photothermal effects and enables efficient intracellular delivery of payload molecules into various cell types. By incorporating photothermal NPs within biocompatible electrospun nanofibers, direct cellular contact with NPs is avoided, thus largely mitigating safety or regulatory issues. Importantly, PEN photoporation is gentler to cells compared with electroporation, the most commonly used physical transfection method, resulting in higher-quality genetically engineered cells with better therapeutic potential. According to this protocol, it takes 2-3 d to prepare PEN culture wells with the desired cells, 3-4 d to optimize PEN photoporation parameters for intracellular delivery of payload molecules into different cell types in vitro and 4-5 weeks to evaluate the in vivo therapeutic efficacy of PEN-photoporated T cells. The protocol also provides details on how to construct the laser-based setup for performing photoporation experiments.

Substrate stiffness dictates unique doxorubicin-induced senescence-associated secretory phenotypes and transcriptomic signatures in human pulmonary fibroblasts.

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-β-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2kPa), fibrotic (50kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21 + cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

AbstractMesenchymal stem cell‐derived extracellular vesicles (MSC EVs) are an attractive therapeutic option for regenerative medicine applications due to their inherently pro‐angiogenic and anti‐inflammatory properties. However, reproducible and cost‐effective production of highly potent therapeutic MSC EVs is challenging, limiting their translational potential. Here, we investigated whether the well‐characterized responsiveness of MSCs to their mechanical environment—specifically, substrate stiffness—could be exploited to generate EVs with increased therapeutic bioactivity without the need for biochemical priming or genetic manipulation. Using polydimethylsiloxane and bone marrow‐derived MSCs (BM‐MSCs), we show that decreasing the stiffness of MSC substrates to as low as 3 kPa significantly improves the pro‐angiogenic bioactivity of EVs as measured by tube formation and gap closure assays. We also demonstrate that lower substrate stiffness improves EV production and overall yield, important for clinical translation. Furthermore, we establish the mechanoresponsiveness of induced pluripotent stem cell‐derived MSC (iMSC) EVs and their comparability to BM‐MSC EVs, again using tube formation and gap closure assays. With this data, we confirm iMSCs' feasibility as an alternative, renewable cell source for EV production with reduced donor variability. Overall, these results suggest that utilizing substrate stiffness is a promising, simple, and a potentially scalable approach that does not require exogenous cargo or extraneous reagents to generate highly potent pro‐angiogenic MSC EVs.

Biomaterial-based strategies for primary human corneal endothelial cells for therapeutic applications: from cell expansion to transplantable carrier.

The treatment of corneal blindness due to corneal diseases and injuries often requires the transplantation of healthy cadaveric corneal endothelial graft tissue to restore corneal clarity and visual function. However, the limited availability of donor corneas poses a significant challenge in meeting the demand for corneal transplantation. As a result, there is a growing interest in developing strategies alleviate this unmet need, and one of the postulated approaches is to isolate and expand primary human corneal endothelial cells (HCECs) in vitro for use in cell therapy. This review summarizes the recent advancements in the expansion of HCECs using biomaterials. Two principal biomaterial-based approaches, including extracellular matrix (ECM) coating and functionalized synthetic polymers, have been investigated to create an optimal microenvironment for the expansion and maintenance of corneal endothelial cells (CECs). This review highlights the challenges and opportunities in expanding primary HCECs using biomaterials. It emphasizes the importance of optimizing biomaterial properties, cell culture conditions, and the roles of biophysical cues to achieve efficient expansion and functional maintenance of CECs. Biomaterial-based strategies hold significant promise for expanding primary HCECs and improving the outcomes of CEC transplantation. The integration of biomaterials as cell culture substrates and transplantable carriers offers a comprehensive approach to address the limitations associated with current corneal tissue engineering techniques.

Direct activation of Toll-like receptor 2 signaling stimulated by contact with the interfacial structures of chitin nanofibers

The innate immune system, which eliminates pathogens and abnormal cells, is involved in the pathogenesis of various diseases and infections, where Toll-like receptors (TLRs) play a critical regulatory role. In this study, we investigated the potential of chitin nanofiber (CtNF) to induce an immune response, which is expected to act as an agonist of TLR2. Crab-derived CtNF, surface-deacetylated CtNF, and surface-carboxylated cellulose NF were employed as TLR2-mediated immune stimulator, signal regulator, and cell adhesion promoter, respectively, to fabricate cell culture scaffolds for HEK293 cells with TLR2 and human monocyte THP-1 cells with or without TLR2. Surface deacetylation of CtNF drastically diminished the immunological response of HEK293 cells, suggesting that the N-acetyl groups on the solid CtNF surface were pivotal for TLR2-mediated stimulation. A comparison of wild-type and TLR2-KO THP-1 cells on cell culture substrates with N-acetyl groups ranging from 0 to 1.39 mmol g−1 revealed that immune signaling for nuclear factor-κB and interferon regulatory factor pathways was strongly dependent on the surface N-acetyl group content. The immunostimulatory level at the interface of solid CtNF and immune cells could be regulated by simply mixing CtNF and surface-deacetylated CtNF, which is a significant advantage for its potential use as a novel immunostimulant.

Graphene SU-8 Platform for Enhanced Cardiomyocyte Maturation and Intercellular Communication in Cardiac Drug Screening.

Cell culture substrates designed for myocardial applications are pivotal in promoting the maturation and functional integration of cardiomyocytes. However, traditional in vitro models often inadequately mimic the diverse biochemical signals and electrophysiological properties of mature cardiomyocytes. Herein, we propose the application of monolayer graphene, transferred onto SU-8 cantilevers integrated with a microelectrode array, to evaluate its influence on the structural, functional, and electro-mechano-physiological properties of cardiomyocytes. The monolayer graphene, prepared using chemical vapor deposition, is adeptly transferred to the target substrates via thermal release tape. The electrical conductivity of these graphene-enhanced SU-8 substrates is about 1600 S/cm, markedly surpassing that of previously reported cell culture substrates. Immunofluorescence staining and Western blot analyses reveal that the electrically conductive graphene significantly enhances cardiomyocyte maturation and cardiac marker expression compared to bare SU-8 substrates. Cardiomyocytes cultured on graphene-transferred substrates exhibit conduction velocity approximately 3.4 times greater than that of the control group. Such improvements in cardiac marker expression, mechano-electrophysiological performance lead to better responsiveness to cardiovascular drugs, such as Verapamil and Isoproterenol. While the graphene monolayer does not fully replicate the complex environment found in native cardiac tissue, its use on SU-8 substrates offers a feasible approach for accelerating cardiomyocyte maturation and facilitating drug screening applications.

Development of Biomimetic Substrates for Limbal Epithelial Stem Cells Using Collagen-Based Films, Hyaluronic Acid, Immortalized Cells, and Macromolecular Crowding.

Despite the promising potential of cell-based therapies developed using tissue engineering techniques to treat a wide range of diseases, including limbal stem cell deficiency (LSCD), which leads to corneal blindness, their commercialization remains constrained. This is primarily attributable to the limited cell sources, the use of non-standardizable, unscalable, and unsustainable techniques, and the extended manufacturing processes required to produce transplantable tissue-like surrogates. Herein, we present the first demonstration of the potential of a novel approach combining collagen films (CF), hyaluronic acid (HA), human telomerase-immortalized limbal epithelial stem cells (T-LESCs), and macromolecular crowding (MMC) to develop innovative biomimetic substrates for limbal epithelial stem cells (LESCs). The initial step involved the fabrication and characterization of CF and CF enriched with HA (CF-HA). Subsequently, T-LESCs were seeded on CF, CF-HA, and tissue culture plastic (TCP). Thereafter, the effect of these matrices on basic cellular function and tissue-specific extracellular matrix (ECM) deposition with or without MMC was evaluated. The viability and metabolic activity of cells cultured on CF, CF-HA, and TCP were found to be similar, while CF-HA induced the highest (p < 0.05) cell proliferation. It is notable that CF and HA induced cell growth, whereas MMC increased (p < 0.05) the deposition of collagen IV, fibronectin, and laminin in the T-LESC culture. The data highlight the potential of, in particular, immortalized cells and MMC for the development of biomimetic cell culture substrates, which could be utilized in ocular surface reconstruction following further in vitro, in vivo, and clinical validation of the approach.

Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts.

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-β-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2 kPa), fibrotic (50 kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21+ cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

The Photocleavable Protein PhoCl-Based Dynamic Hydrogels.

Dynamic protein hydrogels have attracted increasing attention owing to their tunable physiochemical and mechanical properties, customized functionality, and biocompatibility. Among the different types of dynamic hydrogels, photoresponsive hydrogels are of particular interest. Here, we report the engineering of a photoresponsive protein hydrogel by using the photocleavable protein PhoCl. We employed the well-developed SpyTag and SpyCatcher chemistry to engineer PhoCl-containing covalently cross-linked hydrogels. In the hydrogel network, PhoCl, which can be cleaved into two fragments upon violet irradiation, is employed as a dynamic structural motif to regulate the cross-linking density of the hydrogel network. The resultant PhoCl-containing hydrogels showed photoresponsive viscoelastic properties. Upon violet irradiation, the PhoCl hydrogels soften, leading to an irreversible reduction in the storage moduli. However, no gel-sol transition was observed. Leveraging this light-induced stiffness change, we employed this hydrogel as a cell culture substrate to investigate the mechanobiological response of NIH-3T3 fibroblast cells. Our results showed that 3T3 cells can change their morphologies in response to the stiffness change of the PhoCl hydrogel substrate dynamically, rendering PhoCl-based hydrogels a useful substrate for other mechanobiological studies.

Biosensor for integrin inhibition of mammalian cell adhesion and migration using micropatterned cell culture substrate and retroreflective optical signaling

Integrins are a family of transmembrane receptors that play a crucial role in cell adhesion and migration. Integrins can uniquely transduce biochemical signals bidirectionally across the membrane and physically link the cell-cell and cell-extracellular matrix (ECM) with ligand bonds. The arginyl-glycyl-aspartic acid (RGD) peptide motif is present in the ECM as a minimal recognition sequence for integrins. To leverage this property in cell-based therapy, RGD variants, such as cyclic-type RGDfK (c(RGDfK)), which share a similar structure with RGD but exhibit a higher affinity for integrins, have been developed. However, because most evaluation methods for newly developed RGD variants focus on affinity strength, tools for cellular effects are required. In this study, we developed a new platform that integrates micropatterned three-dimensional cell culture substrates with a non-spectroscopic optical analysis system to quantitatively analyze the effects of RGD variants on cell adhesion and migration. The specially micropatterned substrate provides a cell adhesive and migration area to provide a restricted analysis area. Owing to the characteristics of retroreflective Janus particles (RJPs), a non-spectroscopic optical analysis system provides long-term stable optical verification properties and a simple optical setup. These techniques were integrated to quantitatively determine the integrin inhibitory effect of various concentrations of RGD variant. To demonstrate the efficacy of the developed cellular level RGD variant testing platform, the model cell line L929 fibroblast and model RGD variant c(RGDfK) were analyzed ranging from 0 to 10 μM. The results showed that the developed system could effectively and quantitatively analyze the effects of RGD variants on cells across various concentrations.

Transformative Biomaterials in Burn Care: Utilizing Scaffolds, Hydrogels, and Chitosan for Optimized Healing and Scar Reduction

With over a million patients per year in the USA alone, burns are among the most common injuries in the world. In addition to preventing difficulties during burn injuries, the growing impact of scaffolds, hydrogels, cell scaffolds, and cell sheets with healing boosting elements expedite triggers and strengthens re-epithelialization and wound healing, which limits the creation of scars. While superficial partial-thickness and superficial burns typically heal without surgery, severe burns require special care, including topical antimicrobial surgery or bandages. Usually composed of polymeric biomaterials, scaffolds offer the structural support needed for cell adhesion and the subsequent formation of tissue. The term "tissue engineering triad" often refers to cells, scaffolds, and growth-stimulating signals. This may be accomplished in several ways by combining different polymers. Simple (planar) 3D structures may be produced using conventional and rapid prototyping techniques, but complicated structures have been successfully produced by carefully controlling the Molds and processing parameters. For biomedical and tissue engineering applications, chitosan has been widely employed (skin, bone, cartilage, and vascular grafts to substrates for mammalian cell culture). Additionally, it is renewable, biocompatible, biodegradable, nonantigenic, bioactive, and nontoxic.

Thermoresponsive Brush Coatings for Cell Sheet Engineering with Low Protein Adsorption above the Polymers' Phase Transition Temperature.

Thermoresponsive polymer coatings on cell culture substrates enable noninvasive cell detachment and cell sheet fabrication for biomedical applications. Optimized coatings should support controlled culture and detachment of various cell types and allow chemical modifications, e.g., to introduce specific growth factors for enhanced gene expression. Furthermore, the sterilization and storage stability of the coatings must be assessed for translational attempts. Poly(glycidyl ether) (PGE) brush coatings with short alkoxy side chains provide a versatile platform for cell culture and detachment, but their polyether backbones are susceptible to oxidation and degradation. Thus, we rationally designed potential alternatives with thermoresponsive glycerol-based block copolymers comprising a stable polyacrylate or polymethacrylate backbone and an oligomeric benzophenone (BP)-based anchor. The resulting poly(ethoxy hydroxypropyl acrylate-b-benzophenone acrylate) (pEHPA-b-BP) and poly(ethoxy hydroxypropyl methacrylate-b-benzophenone methacrylate) (pEHPMA-b-BP) block copolymers preserve the short alkoxy-terminated side chains of the PGE derived structure on a stable, but hydrophobic, aliphatic backbone. The amphiphilicity balance is maintained through incorporated hydroxyl groups, which simultaneously can be used for chemical modification. The polymers were tailored into brush coatings on polystyrene surfaces via directed adsorption using the BP oligomer anchor. The resulting coatings with thickness values up to ∼3 nm supported efficient adhesion and proliferation of human fibroblasts despite minimal protein adsorption. The conditions for cell sheet fabrication on pEHPA-b-BP were gentler and more reliable than on pEHPMA-b-BP, which required additional cooling. Hence, the stability of pEHPA-b-BP and PGE coatings was evaluated post gamma and formaldehyde (FO) gas sterilization. Gamma sterilization partially degraded PGE coatings and hindered cell detachment on pEHPA-b-BP. In contrast, FO sterilization only slowed detachment on PGE coatings and had no adverse effects on pEHPA-b-BP, maintaining their efficient performance in cell sheet fabrication.

Effects of Clump Size on the Pluripotency and Proliferation in the Passaging Process of Mouse Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are a promising cell source because of their pluripotency and self-renewal abilities. However, there is a risk of pluripotency loss during cell expansion. Particularly, cell passaging is associated with a higher risk of decreasing cell quality. There are two iPSC passaging methods: single-cell and clump passaging. Single-cell passaging is a rapid and simple method for cell manipulation, whereas clump passaging is superior for maintaining iPSC pluripotency. Therefore, clump passaging is a robust method for expanding iPSCs while maintaining their pluripotency. However, clump size control during clump passaging is difficult because colony fragmentation is performed manually by pipetting the colonies detached from the cell culture substrates. In this study, the effect of pipetting on iPSC colony fragmentation was evaluated and the relationship between iPSC clump size and pluripotency was clarified. An automated pipetting device was developed to standardize the clump passage process. The effect of clump size on the pluripotency and proliferative capacity of mouse iPSCs was investigated. Clump size was controlled by varying the number of pipetting cycles, and pluripotency and proliferation were assessed via alkaline phosphatase staining and flow cytometry. Our results revealed that a decrease in clump size corresponded to an increase in cell proliferation, while pluripotency maintenance was optimized under specific clump sizes. These results underscore the significance of clump size for stem cell quality, emphasizing the need for a balanced approach to maintain pluripotency while fostering proliferation in the cell expansion culture for iPSCs.

Using Polycaprolactone Nanofibers for the Proof-of-Concept Construction of the Alveolar-Capillary Interface.

The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface invitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.

Rescue of mitochondrial dysfunction through alteration of extracellular matrix composition in barth syndrome cardiac fibroblasts

Fibroblast-ECM (dys)regulation is associated with a plethora of diseases. The ECM acts as a reservoir of inflammatory factors and cytokines that mediate molecular mechanisms within cardiac cell populations. The role of ECM-mitochondria crosstalk in the development and progression of cardiac disorders remains uncertain. We evaluated the influence of ECM produced by stromal cells from patients with the mitochondrial cardiomyopathy (Barth syndrome, BTHS) and unaffected healthy controls on cardiac fibroblast (CF) metabolic function. To do this, cell-derived matrices CDMs were generated from BTHS and healthy human pluripotent stem cell-derived CFs (hPSC-CF) and used as cell culture substrates. BTHS CDMs negatively impacted the mitochondrial function of healthy hPSC-CFs while healthy CDMs improved mitochondrial function in BTHS hPSC-CFs. Mass spectrometry comparisons identified 5 matrisome proteins differentially expressed in BTHS compared to healthy CDM. Our results highlight a key role for the ECM in disease through its impact on mitochondrial function.

Substrate stiffness-dependent activation of Hippo pathway in cancer associated fibroblasts

The tumor microenvironment (TME) comprises a heterogenous cell population within a complex three-dimensional (3D) extracellular matrix (ECM). Stromal cells within this TME are altered by signaling cues from cancer cells to support uncontrolled tumor growth and invasion events. Moreover, the ECM also plays a fundamental role in tumor development through pathological remodeling, stiffening and interaction with TME cells. In healthy tissues, Hippo signaling pathway actively contributes to tissue growth, cell proliferation and apoptosis. However, in cancer, the Hippo signaling pathway is highly dysregulated, leading to nuclear translocation of the YAP/TAZ complex, which directly contributes to uncontrolled cell proliferation and tissue growth, and ECM remodeling and stiffening processes. Here, we compare the effect of increasing cell culture substrate stiffness, derived from tumor progression, upon the dysregulation of the Hippo signaling pathway in colorectal cancer-associated fibroblasts (CAFs) and normal colorectal fibroblasts (NFs). We correlate the dysregulation of Hippo pathway with the magnitude of the traction forces exerted by healthy and malignant stromal cells. We found that ECM stiffening is crucial in Hippo pathway dysregulation in CAFs, but not in normal fibroblasts.

A Scalable Method to Fabricate 2D Hydrogel Substrates for Mechanobiology Studies with Independent Tuning of Adhesiveness and Stiffness.

Mechanical signals from the extracellular matrix are crucial in guiding cellular behavior. Two-dimensional hydrogel substrates for cell cultures serve as exceptional tools for mechanobiology studies because they mimic the biomechanical and adhesive characteristics of natural environments. However, the interdisciplinary knowledge required to synthetize and manipulate these biomaterials typically restricts their widespread use in biological laboratories, which may not have the material science expertise or specialized instrumentation. To address this, we propose a scalable method that requires minimal setup to produce 2D hydrogel substrates with independent modulation of the rigidity and adhesiveness within the range typical of natural tissues. In this method, norbornene-terminated 8-arm polyethylene glycol is stoichiometrically functionalized with RGD peptides and crosslinked with a di-cysteine terminated peptide via a thiol-ene click reaction. Since the synthesis process significantly influences the final properties of the hydrogels, we provide a detailed description of the chemical procedure to ensure reproducibility and high throughput results. We demonstrate examples of cell mechanosignaling by monitoring the activation state of the mechanoeffector proteins YAP/TAZ. This method effectively dissects the influence of biophysical and adhesive cues on cell behavior. We believe that our procedure will be easily adopted by other cell biology laboratories, improving its accessibility and practical application.

A Novel Recombinant Vitronectin Variant Supports the Expansion and Differentiation of Pluripotent Stem Cells in Defined Animal-Free Workflows.

An essential aspect of harnessing the potential of pluripotent stem cells (PSCs) and their derivatives for regenerative medicine is the development of animal-free and chemically defined conditions for ex vivo cultivation. PSCs, including embryonic and induced PSCs (iPSCs), are in the early stages of clinical trials for various indications, including degenerative diseases and traumatic injury. A key step in the workflows generating these cells for more widespread clinical use is their safe and robust ex vivo cultivation. This entails optimization of cell culture media and substrates that are safe and consistent while maintaining robust functionality. Here, we describe the design of a human vitronectin (hVTN) variant with improved manufacturability in a bacterial expression system along with improved function in comparison to wild-type VTN and other previously characterized polypeptide fragments. In conjunction with an animal component-free media formulation, our hVTN fragment provides animal-free conditions for the enhanced expansion of iPSCs. This hVTN variant also supports the reprogramming of PBMCs into iPSCs. Furthermore, we show that these iPSCs can be efficiently differentiated into the three major germ layers and cortical neurons, thereby closing the loop on a completely defined animal-free workflow for cell types relevant for regenerative medicine.

Collagen as Bioink for Bioprinting: A Comprehensive Review.

Biomaterials made using collagen are successfully used as a three-dimensional (3D) substrate for cell culture and considered to be promising scaffolds for creating artificial tissues. An important task that arises for engineering such materials is the simulation of physical and morphological properties of tissues, which must be restored or replaced. Modern additive technologies, including 3D bioprinting, can be applied to successfully solve this task. This review provides the latest evidence on advances of 3D bioprinting with collagen in the field of tissue engineering. It contains modern approaches for printing pure collagen bioinks consisting only of collagen and cells, as well as the obtained results from the use of pure collagen bioinks in different fields of tissue engineering.

Digital Photoinduced Topographical Microsculpting of Hydrogels

AbstractHydrogels are soft materials widely used as a substrate or matrix for cell culture to mimic the physicochemical, structural, and mechanical properties of the cell's native microenvironment. Recently, a growing body of evidence has shown that substrate topography is a key determinant of cell behavior. These studies, however, mostly rely on the use of hard elastomeric materials, particularly as introducing topography in soft hydrogels is technically challenging, labor intensive, and often does not provide a flexible design space. Here, a new approach is presented that allows robust creation of topographies on hydrogels using a maskless and contactless UV‐sculpting approach. The sculpted topographies are highly programmable: the features can be designed using any digital drawing software, while the dimensions and resolution can be easily tuned via UV exposure dose or photoinitiator concentration. Microscale topographies (2–100 µm in size) can be created on a wide range of hydrogels with diverse chemical characters and mechanical properties (e.g., stiffness). As a proof of concept, this work shows that adherent cells on softer hydrogels align better to topographical cues than on stiffer hydrogels, underlining the importance of studying cell behavior in physiologically more relevant multi‐cue environments.