Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 1.935
Filter
1.
Metabolomics ; 20(3): 44, 2024 Apr 06.
Article in English | MEDLINE | ID: mdl-38581549

ABSTRACT

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.


Subject(s)
Hypothermia , Humans , Organ Preservation/methods , Metabolomics , Cornea , Organ Culture Techniques/methods
2.
J Vis Exp ; (206)2024 Apr 12.
Article in English | MEDLINE | ID: mdl-38682940

ABSTRACT

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs. Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (≥30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.


Subject(s)
Neural Stem Cells , Spinal Cord Injuries , Spinal Cord , Animals , Mice , Spinal Cord Injuries/therapy , Humans , Neural Stem Cells/cytology , Neural Stem Cells/transplantation , Spinal Cord/cytology , Organ Culture Techniques/methods , Stem Cell Transplantation/methods
3.
J Neurosci Methods ; 404: 110055, 2024 04.
Article in English | MEDLINE | ID: mdl-38184112

ABSTRACT

The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. Especially brain slices have proven to be an excellent source to investigate brain physiology and disease at cellular and small network level, overcoming the temporal limits of acute slices. Here we provide a revised, detailed protocol of the production and in-depth knowledge on long-term culturing of such human organotypic brain slice cultures for research purposes. We highlight the critical pitfalls of the culturing process of the human brain tissue and present exemplary results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings as readouts for culture viability, enabling the use of organotypic brain slice cultures of these valuable tissue samples for basic neuroscience and disease modeling (Fig. 1).


Subject(s)
Brain , Neurons , Humans , Brain/metabolism , Neurons/physiology , Electrodes , Organ Culture Techniques/methods
4.
J Appl Toxicol ; 44(5): 784-793, 2024 05.
Article in English | MEDLINE | ID: mdl-38262615

ABSTRACT

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.


Subject(s)
Cisplatin , Testis , Humans , Mice , Animals , Child , Infant, Newborn , Male , Testis/metabolism , Organ Culture Techniques/methods , Cisplatin/toxicity , Spermatogenesis , Green Fluorescent Proteins/genetics
5.
Methods Mol Biol ; 2749: 91-101, 2024.
Article in English | MEDLINE | ID: mdl-38133777

ABSTRACT

Models have been extensively used to investigate disease pathogenesis. Animal models are costly and require extensive logistics for animal care, and samples are not always suitable for different analytical techniques or to answer the research question. In vitro cell culture models are generally focused on recreating a specific characteristic of an organ and are limited to a single cell population that does not display the characteristic tissue architecture of the source organ. In addition, such models do not account for the many interactions between pathogens and the diverse cell subsets that are normally present in a given organ. Conclusions based on conventional 2D cell culture methods are limited, requiring extrapolation from a reductionist model to understand in vivo events. In vitro organ culture (IVOC) offers a way to overcome some of these limitations. Explants conserve important in vivo characteristics, such as different cell types and complex tissue architecture. This in vitro (ex vivo) organ culture protocol of the swine large intestine aims at maintaining viable colonic mucosa for up to 5 days. The protocol described herein applies a combination of methods used for immortalized cell culture and stem cell stimulation to support the physiological cellular flow inherent of the intestinal mucosa. Required equipment includes a hyperoxic chamber and culture at the air-liquid interface.


Subject(s)
Colon , Intestinal Mucosa , Swine , Animals , Organ Culture Techniques/methods , Cell Culture Techniques , Models, Animal
6.
J Virol ; 96(7): e0009822, 2022 04 13.
Article in English | MEDLINE | ID: mdl-35285684

ABSTRACT

Respiratory viruses cause mild to severe diseases in humans every year, constituting a major public health problem. Characterizing the pathogenesis in physiologically relevant models is crucial for developing efficient vaccines and therapeutics. Here, we show that lung organoids derived from human primary or lung tumor tissue maintain the cellular composition and characteristics of the original tissue. Moreover, we show that these organoids sustain viral replication with particular infection foci formation, and they activate the expression of interferon-associated and proinflammatory genes responsible for mediating a robust innate immune response. All together, we show that three-dimensional (3D) lung organoids constitute a relevant platform to model diseases and enable the development of drug screenings. IMPORTANCE Three-dimensional (3D) human lung organoids reflect the native cell composition of the lung as well as its physiological properties. Human 3D lung organoids offer ideal conditions, such as timely availability in large quantities and high physiological relevance for reassessment and prediction of disease outbreaks of respiratory pathogens and pathogens that use the lung as a primary entry portal. Human lung organoids can be used in basic research and diagnostic settings as early warning cell culture systems and also serve as a relevant platform for modeling infectious diseases and drug development. They can be used to characterize pathogens and analyze the influence of infection on, for example, immunological parameters, such as the expression of interferon-associated and proinflammatory genes in the context of cancer. In our study, we found that cancer-derived lung organoids were more sensitive to influenza A virus infection than those derived from healthy tissue and demonstrated a decreased innate immune response.


Subject(s)
Lung , Organ Culture Techniques , Organoids , Communicable Diseases/diagnosis , Communicable Diseases/immunology , Humans , Immunity, Innate , Interferons , Lung/pathology , Organ Culture Techniques/methods , Organoids/immunology , Organoids/virology
7.
Nat Protoc ; 17(1): 15-35, 2022 01.
Article in English | MEDLINE | ID: mdl-34992269

ABSTRACT

The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.


Subject(s)
Brain/cytology , Nerve Net , Neurophysiology/methods , Organoids , Cell Culture Techniques/methods , Cells, Cultured , Humans , Molecular Imaging , Nerve Net/cytology , Nerve Net/diagnostic imaging , Nerve Net/physiology , Optogenetics , Organ Culture Techniques/methods , Organoids/cytology , Organoids/diagnostic imaging , Organoids/physiology , Pluripotent Stem Cells/cytology
8.
Cancer Res ; 82(3): 510-520, 2022 02 01.
Article in English | MEDLINE | ID: mdl-34872965

ABSTRACT

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.


Subject(s)
Biomarkers, Pharmacological/chemistry , Gene Expression/genetics , Microfluidics/methods , Organ Culture Techniques/methods , Humans
9.
Mol Cell Endocrinol ; 539: 111488, 2022 01 01.
Article in English | MEDLINE | ID: mdl-34637880

ABSTRACT

PURPOSE: of the research: To achieve male fertility preservation and restoration, experimental strategies for in vitro germ cell differentiation are required. The effects of two different culture conditions on in vitro maintenance and differentiation of non-human primate germ cells was studied. Three testes from three 6-month-old marmosets were cultured using a gas-liquid interphase system for 12 days. Testicular maturation in pre-culture control and samples cultured in gonadotropin and serum supplemented and non-supplemented culture samples was evaluated using Periodic Acid-Schiff (PAS) and immunohistochemical stainings. PRINCIPLE RESULTS: Gonadotropins and serum-supplemented tissues demonstrate up to meiotic differentiation (BOULE + Pachytene spermatocyte) and advanced localization of germ cells (MAGEA4+). Moreover, complex (with gonadotropin and marmoset monkey serum) conditions induced progression in somatic cell maturation with advanced seminiferous epithelial organization, maintenance of encapsulation of cultured fragments with peritubular-myoid cells, preservation of tubular structural integrity and architecture. MAJOR CONCLUSIONS: We report stimulation-dependent in vitro meiotic transition in non-human primate testes. This model represents a novel ex vivo approach to obtain crucial developmental progression.


Subject(s)
Culture Media, Serum-Free/pharmacology , Gonadotropins/pharmacology , Organ Culture Techniques/methods , Testis/cytology , Animals , Callithrix , Cell Differentiation , Male , Meiosis , Sexual Maturation , Spermatogenesis
10.
Int J Mol Sci ; 22(24)2021 Dec 07.
Article in English | MEDLINE | ID: mdl-34947977

ABSTRACT

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.


Subject(s)
Adult Stem Cells/cytology , Organ Culture Techniques/methods , Organoids/cytology , Cell Differentiation , Heart/physiology , Humans , Models, Biological , Pluripotent Stem Cells/cytology , Regeneration , Spheroids, Cellular/cytology
11.
Int J Mol Sci ; 22(24)2021 Dec 08.
Article in English | MEDLINE | ID: mdl-34948015

ABSTRACT

Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.


Subject(s)
Metatarsal Bones/embryology , Metatarsal Bones/growth & development , NF-kappa B/metabolism , Organ Culture Techniques/methods , Aggrecans/genetics , Animals , Biomechanical Phenomena , Collagen Type II/genetics , Culture Media , Gene Expression Profiling , Gene Expression Regulation, Developmental , Hydrogels , Metatarsal Bones/metabolism , Mice , Mice, Inbred C57BL , Signal Transduction , TOR Serine-Threonine Kinases/metabolism
12.
Int J Mol Sci ; 22(23)2021 Dec 05.
Article in English | MEDLINE | ID: mdl-34884959

ABSTRACT

Thoracic pair of mammary glands from steroid hormone-pretreated mice respond to hormones structurally and functionally in organ culture. A short exposure of glands for 24 h to 7,12 Dimethylbenz(a)anthracene (DMBA) during a 24-day culture period induced alveolar or ductal lesions. Methods: To differentiate the functional significance of ERα and ERß, we employed estrogen receptor (ER) knockout mice. We compared the effects of DMBA on the development of preneoplastic lesions in the glands in the absence of ERα (αERKO) and ERß (ßERKO) using an MMOC protocol. Glands were also subjected to microarray analyses. We showed that estradiol can be replaced by EGF for pretreatment of mice. The carcinogen-induced lesions developed under both steroids and EGF pretreatment protocols. The glands from αERKO did not develop any lesions, whereas in ßERKO mice in which ERα is intact, mammary alveolar lesions developed. Comparison of microarrays of control, αERKO and ßERKO mice showed that ERα was largely responsible for proliferation and the MAP kinase pathways, whereas ERß regulated steroid metabolism-related genes. The results indicate that ERα is essential for the development of precancerous lesions. Both subtypes, ERα and Erß, differentially regulated gene expression in mammary glands in organ cultures.


Subject(s)
Anthracenes/adverse effects , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/genetics , Mammary Glands, Animal/cytology , Organ Culture Techniques/methods , Piperidines/adverse effects , Precancerous Conditions/pathology , Animals , Epidermal Growth Factor/administration & dosage , Epidermal Growth Factor/pharmacology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/pathology , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Precancerous Conditions/chemically induced , Precancerous Conditions/genetics , Signal Transduction/drug effects
14.
Sci Rep ; 11(1): 22671, 2021 11 22.
Article in English | MEDLINE | ID: mdl-34811401

ABSTRACT

Microglia are resident immune cells in the central nervous system, showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia's morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated. First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma in the retina. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.


Subject(s)
Chemotaxis/physiology , Microglia/metabolism , Models, Biological , Retina/metabolism , Signal Transduction/physiology , Adenosine Triphosphate/metabolism , Animals , Animals, Newborn , Cell Communication/physiology , Immunohistochemistry/methods , Kinetics , Mice , Mice, Inbred ICR , Organ Culture Techniques/methods , Retina/growth & development
15.
Int J Mol Sci ; 22(21)2021 Nov 05.
Article in English | MEDLINE | ID: mdl-34769409

ABSTRACT

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.


Subject(s)
Adenoviridae/genetics , Genetic Vectors/administration & dosage , Macular Degeneration/pathology , Organ Culture Techniques/methods , Retinal Pigment Epithelium/cytology , Transgenes , Animals , Cells, Cultured , Macular Degeneration/genetics , Macular Degeneration/metabolism , Mice , Mice, Inbred C57BL , Models, Animal , Retinal Pigment Epithelium/metabolism , Transduction, Genetic
16.
Nat Neurosci ; 24(11): 1542-1554, 2021 11.
Article in English | MEDLINE | ID: mdl-34675437

ABSTRACT

Amyotrophic lateral sclerosis overlapping with frontotemporal dementia (ALS/FTD) is a fatal and currently untreatable disease characterized by rapid cognitive decline and paralysis. Elucidating initial cellular pathologies is central to therapeutic target development, but obtaining samples from presymptomatic patients is not feasible. Here, we report the development of a cerebral organoid slice model derived from human induced pluripotent stem cells (iPSCs) that recapitulates mature cortical architecture and displays early molecular pathology of C9ORF72 ALS/FTD. Using a combination of single-cell RNA sequencing and biological assays, we reveal distinct transcriptional, proteostasis and DNA repair disturbances in astroglia and neurons. We show that astroglia display increased levels of the autophagy signaling protein P62 and that deep layer neurons accumulate dipeptide repeat protein poly(GA), DNA damage and undergo nuclear pyknosis that could be pharmacologically rescued by GSK2606414. Thus, patient-specific iPSC-derived cortical organoid slice cultures are a reproducible translational platform to investigate preclinical ALS/FTD mechanisms as well as novel therapeutic approaches.


Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Astrocytes/pathology , Frontotemporal Dementia/pathology , Neurons/pathology , Organoids/pathology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Astrocytes/metabolism , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Neurons/metabolism , Organ Culture Techniques/methods , Organoids/metabolism
17.
Nature ; 599(7884): 268-272, 2021 11.
Article in English | MEDLINE | ID: mdl-34707290

ABSTRACT

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.


Subject(s)
Morphogenesis , Neural Tube/anatomy & histology , Neural Tube/embryology , Organ Culture Techniques/methods , Ectoderm/cytology , Ectoderm/embryology , Humans , Models, Biological , Neural Plate/cytology , Neural Plate/embryology , Neural Tube/cytology , Neural Tube Defects/embryology , Neural Tube Defects/pathology , Regeneration , Stem Cells/cytology
18.
In Vitro Cell Dev Biol Anim ; 57(8): 808-816, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34608569

ABSTRACT

In vitro spermatogenesis can be performed for marine medaka (Oryzias dancena) via whole testis organ cultures, but spermatogenesis could only be maintained during the early phase of culturing, suggesting that the culture conditions can be further optimized. To improve the culture conditions, we examined the effects of culture temperature, basal media, and medium supplements on spermatogonial proliferation levels during whole testis organ culturing by BrdU incorporation assays. Our results show that a 30°C culture temperature negatively affected spermatogonial proliferation compared to 26°C and 28°C and that the use of Dulbecco's Modified Eagle Medium and Minimum Essential Medium α (α-MEM) was more effective for spermatogonial proliferation than the use of Leibovitz's L-15 Medium (L15). When fetal bovine serum (FBS) was replaced with KnockOut Serum Replacement (KSR), a significantly positive effect was observed for the maintenance of spermatogonial proliferation. However, supplementation of the medium with 17α, 20ß-dihydroxy-4-pregnen-3-one did not show any significant effect. Gene expression analyses of four genes, including Nanos2, SCP3, AMH, and StAR, indicated that the optimized culture conditions consisting of α-MEM and KSR had the most positive influence on the maintenance of spermatogonial proliferation levels in whole testis organ cultures compared to the original culture conditions consisting of L15 and FBS by maintaining the function of Sertoli and Leydig cells. The results from this study will provide useful information for the study of in vitro spermatogenesis in fish.


Subject(s)
Organ Culture Techniques/methods , Oryzias/physiology , Spermatogenesis/physiology , Testis/physiology , Animals , Culture Media , Male , Temperature
19.
Stem Cell Reports ; 16(9): 2076-2077, 2021 09 14.
Article in English | MEDLINE | ID: mdl-34525383

ABSTRACT

The European Commission Joint Research Centre and the European Standardization Organizations CEN and CENELEC organized the "Putting Science into Standards" workshop, focusing on organ-on-chip technologies. The workshop, held online on 28-29 April, 2021, aimed at identifying needs and priorities for standards development and suggesting possible ways forward.


Subject(s)
Lab-On-A-Chip Devices/standards , Organ Culture Techniques/standards , Humans , Organ Culture Techniques/methods
20.
STAR Protoc ; 2(3): 100765, 2021 09 17.
Article in English | MEDLINE | ID: mdl-34485937

ABSTRACT

3D cultures of mammary epithelial cells purified from murine models provide a unique resource to study genetically defined breast cancer and response to targeted therapies. Here, we describe step-by-step experimental procedures for the successful establishment of murine mammary organoid lines isolated from mammary glands or mammary tumors driven by mutations in components of the PI3K pathway. These detailed protocols also include procedures to perform assays that can be adopted to screen response to drug treatments and to inform better therapies. For details on potential applications and use of this protocol, please refer to Yip et al. (2020).


Subject(s)
Mammary Glands, Animal/cytology , Mammary Neoplasms, Experimental/pathology , Organ Culture Techniques/methods , Organoids , Phosphatidylinositol 3-Kinases/genetics , Animals , Cell Death/physiology , Cryopreservation , Female , Mammary Glands, Animal/physiology , Mammary Neoplasms, Experimental/genetics , Metabolic Networks and Pathways , Mice, Inbred C57BL , Organ Culture Techniques/instrumentation , Phosphatidylinositol 3-Kinases/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...