Cell tumbling enhances stem cell differentiation in hydrogels via nuclear mechanotransduction.

Cells can deform their local niche in three dimensions via whole-cell movements such as spreading, migration or volume expansion. These behaviours, occurring over hours to days, influence long-term cell fates including differentiation. Here we report a whole-cell movement that occurs in sliding hydrogels at the minutes timescale, termed cell tumbling, characterized by three-dimensional cell dynamics and hydrogel deformation elicited by heightened seconds-to-minutes-scale cytoskeletal and nuclear activity. Studies inhibiting or promoting the cell tumbling of mesenchymal stem cells show that this behaviour enhances differentiation into chondrocytes. Further, it is associated with a decrease in global chromatin accessibility, which is required for enhanced differentiation. Cell tumbling also occurs during differentiation into other lineages and its differentiation-enhancing effects are validated in various hydrogel platforms. Our results establish that cell tumbling is an additional regulator of stem cell differentiation, mediated by rapid niche deformation and nuclear mechanotransduction.

In Situ UNIversal Orthogonal Network (UNION) Bioink Deposition for Direct Delivery of Corneal Stromal Stem Cells to Corneal Wounds.

The scarcity of human donor corneal graft tissue worldwide available for corneal transplantation necessitates the development of alternative therapeutic strategies for treating patients with corneal blindness. Corneal stromal stem cells (CSSCs) have the potential to address this global shortage by allowing a single donor cornea to treat multiple patients. To directly deliver CSSCs to corneal defects within an engineered biomatrix, we developed a UNIversal Orthogonal Network (UNION) collagen bioink that crosslinks in situ with a bioorthogonal, covalent chemistry. This cell-gel therapy is optically transparent, stable against contraction forces exerted by CSSCs, and permissive to the efficient growth of corneal epithelial cells. Furthermore, CSSCs remain viable within the UNION collagen gel precursor solution under standard storage and transportation conditions. This approach promoted corneal transparency and re-epithelialization in a rabbit anterior lamellar keratoplasty model, indicating that the UNION collagen bioink serves effectively as an in situ -forming, suture-free therapy for delivering CSSCs to corneal wounds. TEASER. Corneal stem cells are delivered within chemically crosslinked collagen as a transparent, regenerative biomaterial therapy.

The lysogenic filamentous Pseudomonas bacteriophage phage Pf slows mucociliary transport.

Pseudomonas aeruginosa is a major pulmonary pathogen causing chronic pulmonary infections in people with cystic fibrosis (CF). The P. aeruginosa filamentous and lysogenic bacteriophage, Pf phage, is abundant in the airways of many people with CF and has been associated with poor outcomes in a cross-sectional cohort study. Previous studies have identified roles for Pf phage in biofilm formation, specifically forming higher-order birefringent, liquid crystals when in contact with other biopolymers in biofilms. Liquid crystalline biofilms are more adherent and viscous than those without liquid crystals. A key feature of biofilms is to enhance bacterial adherence and resist physical clearance. The effect of Pf phage on mucociliary transport is unknown. We found that primary CF and non-CF nasal epithelial cells cultured at air-liquid interface treated with Pf phage exhibit liquid crystalline structures in the overlying mucus. On these cell cultures, Pf phage entangles cilia but does not affect ciliary beat frequency. In both these in vitro cell cultures and in an ex vivo porcine trachea model, introduction of Pf phage decreases mucociliary transport velocity. Pf phage also blocks the rescue of mucociliary transport by CF transmembrane conductance regulator modulators in CF cultures. Thus, Pf phage may contribute to the pathogenesis of P. aeruginosa-associated CF lung disease via induction of liquid crystalline characteristics to airway secretions, leading to impaired mucociliary transport. Targeting Pf phage may be useful in treatment CF as well as other settings of chronic P. aeruginosa infections.

Interpenetrating networks of fibrillar and amorphous collagen promote cell spreading and hydrogel stability.

Hydrogels composed of collagen, the most abundant protein in the human body, are widely used as scaffolds for tissue engineering due to their ability to support cellular activity. However, collagen hydrogels with encapsulated cells often experience bulk contraction due to cell-generated forces, and conventional strategies to mitigate this undesired deformation often compromise either the fibrillar microstructure or cytocompatibility of the collagen. To support the spreading of encapsulated cells while preserving the structural integrity of the gels, we present an interpenetrating network (IPN) of two distinct collagen networks with different crosslinking mechanisms and microstructures. First, a physically self-assembled collagen network preserves the fibrillar microstructure and enables the spreading of encapsulated human corneal mesenchymal stromal cells. Second, an amorphous collagen network covalently crosslinked with bioorthogonal chemistry fills the voids between fibrils and stabilizes the gel against cell-induced contraction. This collagen IPN balances the biofunctionality of natural collagen with the stability of covalently crosslinked, engineered polymers. Taken together, these data represent a new avenue for maintaining both the fiber-induced spreading of cells and the structural integrity of collagen hydrogels by leveraging an IPN of fibrillar and amorphous collagen networks. Statement of significance: Collagen hydrogels are widely used as scaffolds for tissue engineering due to their support of cellular activity. However, collagen hydrogels often undergo undesired changes in size and shape due to cell-generated forces, and conventional strategies to mitigate this deformation typically compromise either the fibrillar microstructure or cytocompatibility of the collagen. In this study, we introduce an innovative interpenetrating network (IPN) that combines physically self-assembled, fibrillar collagen-ideal for promoting cell adhesion and spreading-with covalently crosslinked, amorphous collagen-ideal for enhancing bulk hydrogel stability. Our IPN design maintains the native fibrillar structure of collagen while significantly improving resistance against cell-induced contraction, providing a promising solution to enhance the performance and reliability of collagen hydrogels for tissue engineering applications.

Porosity dominates over microgel stiffness for promoting chondrogenesis in zwitterionic granular hydrogels.

Granular hydrogels comprised of jammed, crosslinked microgels offer great potential as biomaterial scaffolds for cell-based therapies, including for cartilage tissue regeneration. As stiffness and porosity of hydrogels affect the phenotype of encapsulated cells and the extent of tissue regeneration, the design of tunable granular hydrogels to control and optimize these parameters is highly desirable. We hypothesized that chondrogenesis could be modulated using a granular hydrogel platform based on biocompatible, zwitterionic materials with independent intra- and inter-microgel crosslinking mechanisms. Microgels are made with mechanical fragmentation of photocrosslinked zwitterionic carboxybetaine acrylamide (CBAA) and sulfobetaine methacrylate (SBMA) hydrogels, and secondarily crosslinked in the presence of cells using horseradish peroxide (HRP) to produce cell-laden granular hydrogels. We varied the intra-microgel crosslinking density to produce microgels with varied stiffnesses (1-3 kPa) and swelling properties. These microgels, when resuspended at the same weight fraction and secondarily crosslinked, resulted in granular hydrogels with distinct porosities (5-40%) due to differing swelling properties. The greatest extent of chondrogenesis was achieved in scaffolds with the highest microgel stiffness and highest porosity. However, when scaffold porosity was kept constant and just microgel stiffness varied, cell phenotype and chondrogenesis were similar across scaffolds. These results indicate the dominant role of granular scaffold porosity on chondrogenesis, whereas microgel stiffness appears to play a relatively minor role. These observations are in contrast to cells encapsulated within conventional bulk hydrogels, where stiffness has been shown to significantly affect chondrocyte response. In summary, we introduce chemically-defined, zwitterionic biomaterials to fabricate versatile granular hydrogels allowing for tunable scaffold porosity and microgel stiffness to study and influence chondrogenesis.

Engineered Protein Hydrogels as Biomimetic Cellular Scaffolds.

The biochemical and biophysical properties of the extracellular matrix (ECM) play a pivotal role in regulating cellular behaviors such as proliferation, migration, and differentiation. Engineered protein-based hydrogels, with highly tunable multifunctional properties, have the potential to replicate key features of the native ECM. Formed by self-assembly or crosslinking, engineered protein-based hydrogels can induce a range of cell behaviors through bioactive and functional domains incorporated into the polymer backbone. Using recombinant techniques, the amino acid sequence of the protein backbone can be designed with precise control over the chain-length, folded structure, and cell-interaction sites. In this review, the modular design of engineered protein-based hydrogels from both a molecular- and network-level perspective are discussed, and summarize recent progress and case studies to highlight the diverse strategies used to construct biomimetic scaffolds. This review focuses on amino acid sequences that form structural blocks, bioactive blocks, and stimuli-responsive blocks designed into the protein backbone for highly precise and tunable control of scaffold properties. Both physical and chemical methods to stabilize dynamic protein networks with defined structure and bioactivity for cell culture applications are discussed. Finally, a discussion of future directions of engineered protein-based hydrogels as biomimetic cellular scaffolds is concluded.

In Situ-Forming, Bioorthogonally Cross-linked, Nanocluster-Reinforced Hydrogel for the Regeneration of Corneal Defects.

Corneal defects can lead to stromal scarring and vision loss, which is currently only treatable with a cadaveric corneal transplant. Although in situ-forming hydrogels have been shown to foster regeneration of the cornea in the setting of stromal defects, the cross-linking, biomechanical, and compositional parameters that optimize healing have not yet been established. This, Corneal defects are also almost universally inflamed, and their rapid closure without fibrosis are critical to preserving vision. Here, an in situ forming, bioorthogonally cross-linked, nanocluster (NC)-reinforced collagen and hyaluronic acid hydrogel (NCColHA hydrogel) with enhanced structural integrity and both pro-regenerative and anti-inflammatory effects was developed and tested within a corneal defect model in vivo. The NCs serve as bioorthogonal nanocross-linkers, providing higher cross-linking density than polymer-based alternatives. The NCs also serve as delivery vehicles for prednisolone (PRD) and the hepatocyte growth factor (HGF). NCColHA hydrogels rapidly gel within a few minutes upon administration and exhibit robust rheological properties, excellent transparency, and negligible swelling/deswelling behavior. The hydrogel's biocompatibility and capacity to support cell growth were assessed using primary human corneal epithelial cells. Re-epithelialization on the NCColHA hydrogel was clearly observed in rabbit eyes, both ex vivo and in vivo, with expression of normal epithelial biomarkers, including CD44, CK12, CK14, α-SMA, Tuj-1, and ZO-1, and stratified, multilayered morphology. The applied hydrogel maintained its structural integrity for at least 14 days and remodeled into a transparent stroma by 56 days.

Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

Photoactivated growth factor release from bio-orthogonally crosslinked hydrogels for the regeneration of corneal defects.

In situ-forming hydrogels are an attractive option for corneal regeneration, and the delivery of growth factors from such constructs have the potential to improve re-epithelialization and stromal remodeling. However, challenges persist in controlling the release of therapeutic molecules from hydrogels. Here, an in situ-forming bio-orthogonally crosslinked hydrogel containing growth factors tethered via photocleavable linkages (PC-HACol hydrogel) was developed to accelerate corneal regeneration. Epidermal growth factor (EGF) was conjugated to the hydrogel backbone through photo-cleavable (PC) spacer arms and was released when exposed to mild intensity ultraviolet (UV) light (2-5 mW/cm2, 365 nm). The PC-HACol hydrogel rapidly gelled within a few minutes when applied to corneal defects, with excellent transparency and biocompatibility. After subsequent exposure to UV irradiation, the hydrogel promoted the proliferation and migration of corneal epithelial cells in vitro. The rate of re-epithelialization was positively correlated to the frequency of irradiation, verified through ex vivo rabbit cornea organ culture studies. In an in vivo rat corneal wound healing study, the PC-HACol hydrogel exposed to UV light significantly promoted re-epithelialization, the remodeling of stromal layers, and exhibited significant anti-scarring effects, with minimal α-SMA and robust ALDH3A1 expression. Normal differentiation of the regenerated epithelia after healing was evaluated by expression of the corneal epithelial biomarker, CK12. The remodeled cornea exhibited full recovery of corneal thickness and layer number without hyperplasia of the epithelium.

Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model.

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus.

Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of morbidity and mortality in patients with cystic fibrosis, necessitating treatment with inhaled antibiotics. Pf phage is a filamentous bacteriophage produced by Pa that has been reported to act as a structural element in Pa biofilms. Pf presence has been associated with resistance to antibiotics and poor outcomes in cystic fibrosis, though the underlying mechanisms are unclear. Here, we have investigated how Pf phages and sputum biopolymers impede antibiotic diffusion using human sputum samples and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf phages and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf phages in sputum reduce the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf phages and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

Diffusion-Based 3D Bioprinting Strategies.

3D bioprinting has enabled the fabrication of tissue-mimetic constructs with freeform designs that include living cells. In the development of new bioprinting techniques, the controlled use of diffusion has become an emerging strategy to tailor the properties and geometry of printed constructs. Specifically, the diffusion of molecules with specialized functions, including crosslinkers, catalysts, growth factors, or viscosity-modulating agents, across the interface of printed constructs will directly affect material properties such as microstructure, stiffness, and biochemistry, all of which can impact cell phenotype. For example, diffusion-induced gelation is employed to generate constructs with multiple materials, dynamic mechanical properties, and perfusable geometries. In general, these diffusion-based bioprinting strategies can be categorized into those based on inward diffusion (i.e., into the printed ink from the surrounding air, solution, or support bath), outward diffusion (i.e., from the printed ink into the surroundings), or diffusion within the printed construct (i.e., from one zone to another). This review provides an overview of recent advances in diffusion-based bioprinting strategies, discusses emerging methods to characterize and predict diffusion in bioprinting, and highlights promising next steps in applying diffusion-based strategies to overcome current limitations in biofabrication.

Embedded 3D Bioprinting of Collagen Inks into Microgel Baths to Control Hydrogel Microstructure and Cell Spreading.

Microextrusion-based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. It is hypothesized that interactions between inks and support baths, which are often composed of granular microgels, can be modulated to control the microscopic structure within these macroscopic-printed constructs. Using printed collagen bioinks crosslinked either through physical self-assembly or bioorthogonal covalent chemistry, it is demonstrated that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero-shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity is modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15-50 µm) results in large pores (≈40 µm) that allow human corneal mesenchymal stromal cells (MSCs) to readily spread, while control samples of cast collagen or collagen printed in non-granular support baths do not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

Cell Microencapsulation Within Engineered Hyaluronan Elastin-Like Protein (HELP) Hydrogels.

Three-dimensional cell encapsulation has rendered itself a staple in the tissue engineering field. Using recombinantly engineered, biopolymer-based hydrogels to encapsulate cells is especially promising due to the enhanced control and tunability it affords. Here, we describe in detail the synthesis of our hyaluronan (i.e., hyaluronic acid) and elastin-like protein (HELP) hydrogel system. In addition to validating the efficacy of our synthetic process, we also demonstrate the modularity of the HELP system. Finally, we show that cells can be encapsulated within HELP gels over a range of stiffnesses, exhibit strong viability, and respond to stiffness cues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Elastin-like protein modification with hydrazine Basic Protocol 2: Nuclear magnetic resonance quantification of elastin-like protein modification with hydrazine Basic Protocol 3: Hyaluronic acid-benzaldehyde synthesis Basic Protocol 4: Nuclear magnetic resonance quantification of hyaluronic acid-benzaldehyde Basic Protocol 5: 3D cell encapsulation in hyaluronan elastin-like protein gels.

A Library of Elastin-like Proteins with Tunable Matrix Ligands for In Vitro 3D Neural Cell Culture.

Hydrogels with encapsulated cells have widespread biomedical applications, both as tissue-mimetic 3D cultures in vitro and as tissue-engineered therapies in vivo. Within these hydrogels, the presentation of cell-instructive extracellular matrix (ECM)-derived ligands and matrix stiffness are critical factors known to influence numerous cell behaviors. While individual ECM biopolymers can be blended together to alter the presentation of cell-instructive ligands, this typically results in hydrogels with a range of mechanical properties. Synthetic systems that allow for the facile incorporation and modulation of multiple ligands without modification of matrix mechanics are highly desirable. In the present work, we leverage protein engineering to design a family of xeno-free hydrogels (i.e., devoid of animal-derived components) consisting of recombinant hyaluronan and recombinant elastin-like proteins (ELPs), cross-linked together with dynamic covalent bonds. The ELP components incorporate cell-instructive peptide ligands derived from ECM proteins, including fibronectin (RGD), laminin (IKVAV and YIGSR), collagen (DGEA), and tenascin-C (PLAEIDGIELTY and VFDNFVL). By carefully designing the protein primary sequence, we form 3D hydrogels with defined and tunable concentrations of cell-instructive ligands that have similar matrix mechanics. Utilizing this system, we demonstrate that neurite outgrowth from encapsulated embryonic dorsal root ganglion (DRG) cultures is significantly modified by cell-instructive ligand content. Thus, this library of protein-engineered hydrogels is a cell-compatible system to systematically study cell responses to matrix-derived ligands.

3D printing microporous scaffolds from modular bioinks containing sacrificial, cell-encapsulating microgels.

Microgel-based biomaterials have inherent porosity and are often extrudable, making them well-suited for 3D bioprinting applications. Cells are commonly introduced into these granular inks post-printing using cell infiltration. However, due to slow cell migration speeds, this strategy struggles to achieve depth-independent cell distributions within thick 3D printed geometries. To address this, we leverage granular ink modularity by combining two microgels with distinct functions: (1) structural, UV-crosslinkable microgels made from gelatin methacryloyl (GelMA) and (2) sacrificial, cell-laden microgels made from oxidized alginate (AlgOx). We hypothesize that encapsulating cells within sacrificial AlgOx microgels would enable the simultaneous introduction of void space and release of cells at depths unachievable through cell infiltration alone. Blending the microgels in different ratios produces a family of highly printable GelMA : AlgOx microgel inks with void fractions ranging from 0.03 to 0.35. As expected, void fraction influences the morphology of human umbilical vein endothelial cells (HUVEC) within GelMA : AlgOx inks. Crucially, void fraction does not alter the ideal HUVEC distribution seen throughout the depth of 3D printed samples. This work presents a strategy for fabricating constructs with tunable porosity and depth-independent cell distribution, highlighting the promise of microgel-based inks for 3D bioprinting.

Human enteroids as a tool to study conventional and ultra-high dose rate radiation.

Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.