Organoid Cultures Research Articles

Formation and Long-Term Culture of hiPSC-Derived Sensory Nerve Organoids Using Microfluidic Devices.

Although methods for generating human induced pluripotent stem cell (hiPSC)-derived motor nerve organoids are well established, those for sensory nerve organoids are not. Therefore, this study investigated the feasibility of generating sensory nerve organoids composed of hiPSC-derived sensory neurons using a microfluidic approach. Notably, sensory neuronal axons from neurospheres containing 100,000 cells were unidirectionally elongated to form sensory nerve organoids over 6 mm long axon bundles within 14 days using I-shaped microchannels in microfluidic devices composed of polydimethylsiloxane (PDMS) chips and glass substrates. Additionally, the organoids were successfully cultured for more than 60 days by exchanging the culture medium. The percentage of nuclei located in the distal part of the axon bundles (the region 3-6 mm from the entrance of the microchannel) compared to the total number of cells in the neurosphere was 0.005% for live cells and 0.008% for dead cells. Molecular characterization confirmed the presence of the sensory neuron marker ISL LIM homeobox 1 (ISL1) and the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Moreover, capsaicin stimulation activated TRPV1 in organoids, as evidenced by significant calcium ion influx. Conclusively, this study demonstrated the feasibility of long-term organoid culture and the potential applications of sensory nerve organoids in bioengineered nociceptive sensors.

Deacetylation mimetic mutation of mitochondrial SOD2 attenuates ANG II-induced hypertension by protecting against oxidative stress and inflammation.

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite the use of multiple drugs, likely because of mechanisms that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. Hypertension is associated with inactivation of key intrinsic mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to hyperacetylation, but the role of specific SOD2 lysine residues has not been defined. Hypertension is associated with SOD2 acetylation at lysine 68, and we suggested that deacetylation mimetic mutation of K68 to arginine in SOD2 inhibits vascular oxidative stress and attenuates hypertension. To test this hypothesis, we have developed a new deacetylation mimetic SOD2-K68R mice. We performed in vivo studies in SOD2-K68R mice using angiotensin II (ANG II) model of vascular dysfunction and hypertension. ANG II infusion in wild-type mice induced vascular inflammation and oxidative stress and increased blood pressure to 160 mmHg. SOD2-K68R mutation completely prevented increase in mitochondrial superoxide, abrogated vascular oxidative stress, preserved endothelial nitric oxide production, protected vasorelaxation, and attenuated ANG II-induced hypertension. ANG II and cytokines contribute to vascular oxidative stress and hypertension. Treatment of wild-type aortas with ANG II and cytokines in organoid culture increased mitochondrial superoxide twofold, which was completely prevented in aortas isolated from SOD2-K68R mice. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and pathogenesis of hypertension. We conclude that strategies to reduce SOD2 acetylation may have therapeutic potential in the treatment of vascular dysfunction and hypertension.NEW & NOTEWORTHY Essential hypertension is associated with hyperacetylation of key mitochondrial antioxidant SOD2; however, the pathophysiological role of SOD2 acetylation has not been defined. Our animal study of angiotensin II hypertension model shows that deacetylation mimetic SOD2-K68R mutation prevents pathogenic increase in vascular mitochondrial superoxide, abrogates vascular oxidative stress, preserves endothelial nitric oxide, protects endothelial-dependent vasorelaxation, and attenuates hypertension. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and the pathogenesis of hypertension.

Development of a 3-dimensional organotypic model with characteristics of peripheral sensory nerves

We developed a rat dorsal root ganglion (DRG)-derived sensory nerve organotypic model by culturing DRG explants on an organoid culture device. With this method, a large number of organotypic cultures can be produced simultaneously with high reproducibility simply by seeding DRG explants derived from rat embryos. Unlike previous DRG explant models, this organotypic model consists of a ganglion and an axon bundle with myelinated A fibers, unmyelinated C fibers, and stereo-myelin-forming nodes of Ranvier. The model also exhibits Ca2+ signaling in cell bodies in response to application of chemical stimuli to nerve terminals. Further, axonal transection increases the activating transcription factor 3 mRNA level in ganglia. Axons and myelin are shown to regenerate 14days following transection. Our sensory organotypic model enables analysis of neuronal excitability in response to pain stimuli and tracking of morphological changes in the axon bundle over weeks.

A closed 3D printed microfluidic device for automated growth and differentiation of cerebral organoids from single-cell suspension.

The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.

Establishment and long-term expansion of adult hepatobiliary organoids co-cultured with liver endothelial cells

The liver has a unique ability to regenerate in response to injury or disease with hepatocytes and biliary epithelial cells (BECs) driving the regenerative response. Liver progenitor cells (LPCs) also play role in regeneration with the ability to differentiate into either hepatocytes or BECs. However, during chronic liver disease, the regenerative capacity of the liver is impaired. The use of LPCs is a promising therapeutic strategy for patients with chronic liver diseases. LPCs can be expanded in vitro as self-renewing organoids, however, most approaches to LPC organoids do not include critical cells from the LPC niche in 3D organoid cultures. In this study, we highlight the role of liver endothelial cells (LiECs), as a part of LPC niche, in supporting the hepatobiliary organoids in long-term culture even in the absence of defined growth supplements, such as Wnt agonists. Furthermore, LiECs alter the gene expression profile of hepatobiliary organoids involved in inflammation, migration, extracellular matrix organization, and receptor signaling pathway through paracrine manner. Our findings expand the role of LiECs for regulating stemness of LPCs and elucidate a role for niche cells in a LPC organoid co-culture model with a reduction in growth supplements.

Advancements in 3D bioprinting for precision medicine: Enhancing patient-derived organoids and extracellular vesicle applications in inflammatory diseases

The integration of three-dimensional (3D) bioprinting with cultures of patient-derived organoids represents a transformative advancement in precision medicine, enabling the creation of anatomically accurate and physiologically relevant tissue models. These models are pivotal for personalized disease modeling, therapeutic development, and regenerative medicine. 3D bioprinting enhances the structural and functional fidelity of organoids, thereby improving their application in drug screening and disease intervention strategies. Additionally, this technology facilitates the study of extracellular vesicles (EVs) released from patient-derived organoids (PDOs), which are rich in bioactive molecules and play crucial roles in modulating immune responses. Particularly in chronic inflammatory conditions, these organoid-derived EVs are instrumental in cellular communication and improve our understanding of disease mechanisms beyond traditional biopsies. This review focuses on the latest developments in 3D bioprinting techniques for organoids and EVs, highlighting their potential to revolutionize the treatment of inflammatory diseases through precision medicine.

Peroxisomal cholesterol metabolism regulates yap-signaling, which maintains intestinal epithelial barrier function and is altered in Crohn’s disease

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier, where the cells play essential roles in the digestion of food, absorption of nutrients and water, protection from microbial infections, and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling, which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells, indeed, is linked to gastrointestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and, therefore, homeostasis. Here, we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol, which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover, analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn’s Disease revealed that the dysregulation of peroxisome number, excessive cellular cholesterol, and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn’s Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis.

Patient-derived renal pelvic carcinoma organoids: establishment and sensitivity to chemotherapeutic drugs

Renal pelvic carcinoma is a common upper urothelial cancer. The lack of an ideal in vitro model seriously hinders the research progress in the treatment for this disease. This study established a pipeline for the culture of renal pelvic carcinoma organoids based on the tumor tissue samples derived from the patients and tested the organoids to chemotherapeutic drugs. The results of immunohistochemistry and fluorescence experiments confirmed that the renal pelvic carcinoma organoids obtained from culture presented obvious nuclear heteromorphism, which was consistent with the tissue samples from renal pelvic carcinoma patients. The tumor marker molecule CD44 and the cell proliferation marker molecule Ki67 were positive in the organoids, indicating that the organoids were enriched with tumor stem cells and had strong proliferative ability. The renal pelvic carcinoma organoids were highly sensitive to pirarubicin, which had obvious killing effects. In brief, this study successfully established an in vitro model of renal pelvic cancer organoids and tested the sensitivity of the model to chemotherapeutic drugs. The results provide a new laboratory model for the individualized diagnosis and treatment of epithelial carcinomas represented by renal pelvic carcinoma.

Recent Progress in Generation of Inner Ear Organoid.

Inner ear organoids play a crucial role in hearing research. In comparison to other animal models and 2D cell culture systems, inner ear organoids offer significant advantages for studying the mechanisms of inner ear development and exploring novel approaches to disease treatment. Inner ear organoids derived from human cells are more closely resemble normal human organs in development and function. The 3D culture system of the inner ear organoid enhances cell-cell interactions and mimics the internal environment. In this review, the progress and limitations of organoid culture methods derived from tissue-specific progenitors and pluripotent stem cells (PSCs) are summarized, which may offer new insights into generating organoids that closely resemble the inner ear in terms of morphology and function.

Non-Invasive Quality Control of Organoid Cultures Using Mesofluidic CSTR Bioreactors and High-Content Imaging.

Human brain organoids produce anatomically relevant cellular structures and recapitulate key aspects of in vivo brain function, which holds great potential to model neurological diseases and screen therapeutics. However, the long growth time of 3D systems complicates the culturing of brain organoids and results in heterogeneity across samples hampering their applications. We developed an integrated platform to enable robust and long-term culturing of 3D brain organoids. We designed a mesofluidic bioreactor device based on a reaction-diffusion scaling theory, which achieves robust media exchange for sufficient nutrient delivery in long-term culture. We integrated this device with longitudinal tracking and machine learning-based classification tools to enable non-invasive quality control of live organoids. This integrated platform allows for sample pre-selection for downstream molecular analysis. Transcriptome analyses of organoids revealed that our mesofluidic bioreactor promoted organoid development while reducing cell death. Our platform thus offers a generalizable tool to establish reproducible culture standards for 3D cellular systems for a variety of applications beyond brain organoids.

Chemically Defined Organoid Culture System for Cholangiocyte Differentiation.

Cholangiocyte organoids provide a powerful platform for applications ranging from in vitro modeling to tissue engineering for regenerative medicine. However, their expansion and differentiation are typically conducted in animal-derived hydrogels, which impede the full maturation of organoids into functional cholangiocytes. In addition, these hydrogels are poorly defined and complex, limiting the clinical applicability of organoids. In this study, a novel medium composition combined with synthetic polyisocyanopeptide (PIC) hydrogels to enhance the maturation of intrahepatic cholangiocyte organoids (ICOs) into functional cholangiocytes is utilized. ICOs cultured in the presence of sodium butyrate and valproic acid, a histone deacetylase inhibitor, and a Notch signaling activator, respectively, in PIC hydrogel exhibit a more mature phenotype, as evidenced by increased expression of key cholangiocyte markers, crucial for biliary function. Notably, mature cholangiocyte organoids in PIC hydrogel display apical-out polarity, in contrast to the traditional basal-out polarization of ICOs cultured in Matrigel. Moreover, these mature cholangiocyte organoids effectively model the biliary pro-fibrotic response induced by transforming growth factor beta. Taken together, an animal-free, chemically defined culture system that promotes the ICOs into mature cholangiocytes with apical-out polarity, facilitating regenerative medicine applications and in vitro studies that require access to the apical membrane, is developed.

Establishment of advanced tumor organoids with emerging innovative technologies

Tumor organoids have emerged as a crucial preclinical model for multiple cancer research. Their high establishment rates, stability, and ability to replicate key biological features of original tumor cells in vivo render them invaluable for exploring tumor molecular mechanisms, discovering potential anti-tumor drugs, and predicting clinical drug efficacy. Here, we review the establishment of tumor organoid models and provide an extensive overview of organoid culturing strategies. We also emphasize the significance of integrating cellular components of the tumor microenvironment and physicochemical factors in the organoid culturing system, highlighting the importance of artificial intelligence technology in advancing organoid construction. Moreover, we summarize recent advancements in utilizing organoid systems for novel anti-cancer drug screening and discuss promising trends for enhancing advanced organoids in next-generation disease modeling.

Differentiation of CD166-positive hPSC-derived lung progenitors into airway epithelial cells.

The generation of lung epithelial cells through the directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides a platform to model both embryonic lung development and adult airway disease. Here, we describe a robust differentiation protocol that closely recapitulates human embryonic lung development. Differentiating cells progress through obligate intermediate stages, beginning with definitive endoderm formation and then patterning into anterior foregut endoderm that yields lung progenitors (LPs) with extended culture. These LPs can be purified using the cell surface marker CD166 (also known as ALCAM), and further matured into proximal airway epithelial cells including basal cells, secretory cells and multiciliated cells using either an organoid platform or culture at the air-liquid interface (ALI). We additionally demonstrate that these hPSC-derived airway epithelial cells can be used to model Influenza A infection. Collectively, our results underscore the utility of CD166 expression for the efficient enrichment of LPs from heterogenous differentiation cultures and the ability of these isolated cells to mature into more specialized, physiologically relevant proximal lung cell types.

Exploring cell death mechanisms in spheroid cultures using a novel application of the RIP3-caspase3-assay

This study explores the application of the RIP3-caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay’s capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay’s adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research.

Fully synthetic hydrogels promote robust crypt formation in intestinal organoids.

Initial landmark studies in the design of synthetic hydrogels for intestinal organoid culture identified precise matrix requirements for differentiation, namely decompression of matrix-imposed forces and supplementation of laminin. But beyond stating the necessity of laminin, organoid-laminin interactions have gone largely unstudied, as this ubiquitous requirement of exogenous laminin hinders investigation. In this work, we exploit a fast stress relaxing, boronate ester based synthetic hydrogel for the culture of intestinal organoids, and fortuitously discover that unlike all other synthetic hydrogels to date, laminin does not need to be supplemented for crypt formation. This highly defined material provides a unique opportunity to investigate laminin-organoid interactions and how it influences crypt evolution and organoid function. Via fluorescent labeling of non-canonical amino acids, we further show that adaptable boronate ester bonds increase deposition of nascent proteins, including laminin. Collectively, these results advance the understanding of how mechanical and matricellular signaling influence intestinal organoid development.

Safety assessment of rat embryonic fraction for in vivo regenerative therapy.

Regenerative therapy is considered a novel option for treating various diseases, whereas a developing embryo is a prime source of molecules that help repair diseased tissue and organs. Organoid culture studies also confirmed the inherent biological functions of several embryonic factors. However, the in vivo safety and efficacy of embryonic protein fraction (EPF) were not validated. In this study, we investigated the effectiveness of EPF on healthy adult rats. We obtained embryos from Sprague-Dawley (SD) female rats of E14, E16, and E19 embryonic days and collected protein lysate. This lysate was administered intravenously into adult SD rats on sequential days. We collected blood and performed hematological and biochemical parameters of rats that received EPF. C-reactive protein levels, interleukin-6, blood glucose levels, serum creatinine, blood urea, total leucocyte counts, and % of neutrophils and lymphocytes were comparable between rats receiving EPF and saline. Histological examination of rats' tissues administered with EPF is devoid of abnormalities. Our study revealed that intravenous administration of EPF to healthy adult rats showed that EPF is non-immunogenic, non-inflammatory, non-tumorigenic, and safe for in vivo applications. Our analysis suggests that EPF or its components could be recommended for validating its therapeutic abilities in organ regenerative therapy.

Retroductal dexamethasone administration promotes the recovery from obstructive and inflammatory salivary gland dysfunction.

Salivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models. Functional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone. Dexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1β and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models. Our findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.

The Differential Developmental Neurotoxicity of Valproic Acid on Anterior and Posterior Neural Induction of Human Pluripotent Stem Cells.

Valproic acid (VPA), widely used as an antiepileptic drug, exhibits developmental neurotoxicity when exposure occurs during early or late pregnancy, resulting in various conditions ranging from neural tube defects to autism spectrum disorders. However, toxicity during the very early stages of neural development has not been addressed. Therefore, we investigated the effects of VPA in a model where human pluripotent stem cells differentiate into anterior or posterior neural tissues. Exposure to VPA during the induction of neural stem cells induced different developmental toxic effects in a dose-dependent manner. For instance, VPA induced cell death more profoundly during anteriorly guided neural progenitor induction, while inhibition of cell proliferation and enhanced differentiation were observed during posteriorly guided neural induction. Furthermore, acute exposure to VPA during the posterior induction step also retarded the subsequent neurulation-like tube morphogenesis process in neural organoid culture. These results suggest that VPA exposure during very early embryonic development might exhibit cytotoxicity and subsequently disrupt neural differentiation and morphogenesis processes.

Heparin-Binding Epidermal-like Growth Factor (HB-EGF) Reduces Cell Death in an Organoid Model of Retinal Damage

In zebrafish and various mammalian species, HB-EGF has been shown to promote Müller glia proliferation and activation of repair mechanisms that have not been fully investigated in human retina. In the current study, 70- to 90-day-old human retinal organoids were treated with 20 μM 4-hydroxytamoxifen (4-OHT), and CRX, REC, NRL, PAX6, VIM, GFAP, and VSX2 gene and protein expression were assessed at various times points after treatment. Organoids with or without 4-OHT-induced damage were then cultured with HB-EGF for 7 days. We showed that 20 μM 4-OHT caused a reduction in the number of recoverin-positive cells; an increase in the number of TUNEL-positive cells; and downregulation of the photoreceptor gene markers CRX, NRL, and REC. Culture of organoids with HB-EGF for 7 days after 4-OHT-induced damage caused a marked reduction in the number of TUNEL-positive cells and small increases in the number of Ki67-positive cells and PAX6 and NOTCH1 gene expression. The current results suggest that treatment of human ESC-derived retinal organoids with 4-OHT may be used as a model of retinal degeneration in vitro. Furthermore, HB-EGF treatment of human retinal organoids increases proliferating Müller cells, but only after 4-OHT induced damage, and may be an indication of Muller reactivity in response to photoreceptor damage. Further studies will aim to identify factors that may induce Müller cell-mediated regeneration of the human retina, aiding in the development of therapies for retinal degeneration.

O-096 Human fallopian tube organoids provide a favorable environment for sperm motility

Abstract Study question Does a human fallopian tube (HFT) organoid model offer a favorable apical environment for human sperm survival and motility? Summary answer The apical compartment of a new model of human fallopian tube organoids provides after differentiation a favorable environment for sperm motility. What is known already Human fallopian tubes are the site of major events that are crucial for achieving an ongoing pregnancy, such as gamete survival and competence, fertilization steps, and preimplantation embryo development. In order to better understand the tubal physiology and tubal factors involved in these reproductive functions, and to improve still suboptimal in vitro conditions for gametes preparation and embryo culture during In Vitro Fertilization IVF, we sought to develop a human fallopian tube organoid model from isolated adult stem cells allowing spermatozoa coculture in the apical compartment. Study design, size, duration Over a two-year period, fallopian tube tissues were collected for organoid culture purposes from 10 “donor” patients undergoing bilateral salpingectomy by laparoscopy for definitive sterilization. After tissue digestion, adult stem cells from the isthmus and ampulla regions were separately seeded in 3D Matrigel and cultured with conventional growth factors for organoid culture and specific factors for differentiation of the female genital tract. Participants/materials, setting, methods HFT organoids were characterized by light microscopy, scanning and transmission electron microscopy, immunofluorescence and transcriptomic analysis. Following simultaneous organoid culture on specific inserts, spermatozoa from five donors were placed either in control media or in the apical compartment of colon or HFT organoids (isthmus and ampulla separately) for 96 hours. Vitality and motility were assessed at 0, 48, and 96 hours on 200 spermatozoa in each condition and in duplicate and compared using Wilcoxon test. Main results and the role of chance Specific fallopian tube differentiation of our model was confirmed by immunofluorescence, transcriptome analysis and electron microscopy observations that exhibited ciliary and secretory cells. We succeeded in releasing spermatozoa in the apical compartment of HFT organoids and in recovering them for sperm analysis. Sperm vitality values are similar in HFT organoids and in commercial sperm media. We demonstrated a superiority of HFT organoid apical compartment for sperm motility compared with other controls (colon organoids, organoid culture media, and conventional commercial sperm washing and fertilization media). At 48 hours of incubation, progressive sperm motility was higher in the apical compartment of HFT organoids (ampulla 30.6% ±17.3, isthmus 29.3% ±14.8) than in commercial fertilization media (15.3% ±14.6) (p < 0.05) and compared with all other conditions. At 96 hours progressive sperm motility was almost nil (<1%) in all conditions except for spermatozoa within HFT organoids (p < 0.05): 11.5% ±14.7 and 12.9% ±16.9 in ampulla and isthmus organoids, respectively. Limitations, reasons for caution This was an in vitro study in which conditions of organoid culture could not exactly mimic the in vivo environment of extracellular matrix and the vascularization of fallopian tubes. Wider implications of the findings This work opens up perspectives for better understanding of HFT physiology. For the first time, it highlights the possibility of developing HFT organoids for reproductive purposes. In the future, it could help us to improve gametes fertilizing abilities and culture conditions of embryos during human assisted reproductive technologies. Trial registration number not applicable