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3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction

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A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Reproductive Cells and Development".

Deadline for manuscript submissions: closed (28 February 2022) | Viewed by 36916

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Special Issue Editors

Prof. Dr. Barbara Barboni

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Department of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy
Interests: cell biology; biology of amniotic derived stem cells; tissue regeneration; regenerative medicine; biology of reproduction; ovarian angiogenesis; signal transduction; molecular biology; advanced reproductive biotechnology techniques
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Prof. Dr. Valentina Russo

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Faculty of Bioscience and Agro-Food and Environmental Technology, Agriculture and Environment, University of Teramo, Via Renato Balzarini 1, 64100 Teramo, Italy
Interests: regenerative medicine; tissue engineering; amniotic-derived stem cells
Special Issues, Collections and Topics in MDPI journals

Dr. Nicola Bernabò

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1. Department of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy
2. Institute of Biochemistry and Cell Biology (CNRIBBC/EMMA/Infrafrontier/IMPC), National Research Council, 00015 Rome, Italy
Interests: mammalian reproduction; male physiology; systems biology; electromagnetic fields

Special Issue Information

Dear Colleagues,

We are facing a sort of revolution in biology and biotechnology. The experimental approaches used to explore both physiological and pathological phenomena in vitro and in vivo are very quickly evolving, moving from simple bi-dimensional systems to more complex and reliable set-ups, such as 3D cultures, 3D and 4D printed devices, and organ-on-chip. These new techniques are having increasingly applicative implications in virtually all the fields of biotechnologies and, in particular, in reproductive and regenerative medicine. In this context, in the last decades, the study of reproductive cells, on the one hand, has enormously benefitted by the introduction of these new methods, while on the other hand, it has been considered as a model with which to study different pathologies that represent complex challenges that today's society has to deal with (e.g., reproductive failure, epigenetic diseases, and musculoskeletal tissue regeneration). Most of these pathologies imply an increase in the costs of National Health services, also due to a worsening of citizens’ wellness and the aging of the society.

The application of these innovative techniques, which involve research, clinical approaches, regenerative medicine, and material science, together with the use of the reproductive cells, will provide great advances in human and veterinary medicine contributing to the investigation of both basic and applied issues.

Thus, this Special Issue on “3D cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction” welcomes original research articles that illustrate and stimulate the growing efforts to understand the implication of reproductive cells in physiological conditions, such as developmental biology; embryology; and their application to face pathological environments concerning IVF, ICSI, reproductive disorders, regenerative medicine, and oncology.

Dr. Barbara Barboni
Prof. Valentina Russo
Prof. Dr. Nicola Bernabò
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

reproductive cells
reproductive stem cells
embryology
3D cultures
organoids
organ-on-chip
reproductive medicine
developmental biology
regenerative medicine
tissue engineering

Published Papers (9 papers)

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Research

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21 pages, 3477 KiB

Open AccessEditor’s ChoiceArticle

Hypoxia-Mimetic CoCl₂ Agent Enhances Pro-Angiogenic Activities in Ovine Amniotic Epithelial Cells-Derived Conditioned Medium

by Miriam Di Mattia, Annunziata Mauro, Simona Delle Monache, Fanny Pulcini, Valentina Russo, Paolo Berardinelli, Maria Rita Citeroni, Maura Turriani, Alessia Peserico and Barbara Barboni

Cells 2022, 11(3), 461; https://doi.org/10.3390/cells11030461 - 28 Jan 2022

Cited by 7 | Viewed by 3679

Abstract

Amniotic epithelial stem cells (AECs) are largely studied for their pro-regenerative properties. However, it remains undetermined if low oxygen (O₂) levels that AECs experience in vivo can be of value in maintaining their biological properties after isolation. To this aim, the present study has been designed to evaluate the effects of a hypoxia-mimetic agent, cobalt chloride (CoCl₂), on AECs’ stemness and angiogenic activities. First, a CoCl₂ dose-effect was performed to select the concentration able to induce hypoxia, through HIF-1α stabilization, without promoting any cytotoxicity effect assessed through the analysis of cell vitality, proliferation, and apoptotic-related events. Then, the identified CoCl₂ dose was evaluated on the expression and angiogenic properties of AECs’ stemness markers (OCT-4, NANOG, SOX-2) by analysing VEGF expression, angiogenic chemokines’ profiles, and AEC-derived conditioned media activity through an in vitro angiogenic xeno-assay. Results demonstrated that AECs are sensitive to the cytotoxicity effects of CoCl₂. The unique concentration leading to HIF-1α stabilization and nuclear translocation was 10 µM, preserving cell viability and proliferation up to 48 h. CoCl₂ exposure did not modulate stemness markers in AECs while progressively decreasing VEGF expression. On the contrary, CoCl₂ treatment promoted a significant short-term release of angiogenic chemokines in culture media (CM). The enrichment in bio-active factors was confirmed by the ability of CoCl₂-derived CM to induce HUVEC growth and the cells’ organization in tubule-like structures. These findings demonstrate that an appropriate dose of CoCl₂ can be adopted as a hypoxia-mimetic agent in AECs. The short-term, chemical-induced hypoxic condition can be targeted to enhance AECs’ pro-angiogenic properties by providing a novel approach for stem cell-free therapy protocols. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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30 pages, 6890 KiB

Open AccessFeature PaperArticle

Amniotic Epithelial Stem Cells Counteract Acidic Degradation By-Products of Electrospun PLGA Scaffold by Improving Their Immunomodulatory Profile In Vitro

by Mohammad El Khatib, Valentina Russo, Giuseppe Prencipe, Annunziata Mauro, Ralf Wyrwa, Gabriele Grimm, Miriam Di Mattia, Paolo Berardinelli, Matthias Schnabelrauch and Barbara Barboni

Cells 2021, 10(11), 3221; https://doi.org/10.3390/cells10113221 - 18 Nov 2021

Cited by 7 | Viewed by 2236

Abstract

Electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds with highly aligned fibers (ha-PLGA) represent promising materials in the field of tendon tissue engineering (TE) due to their characteristics in mimicking fibrous extracellular matrix (ECM) of tendon native tissue. Among these properties, scaffold biodegradability must be controlled allowing its replacement by a neo-formed native tendon tissue in a controlled manner. In this study, ha-PLGA were subjected to hydrolytic degradation up to 20 weeks, under di-H₂O and PBS conditions according to ISO 10993-13:2010. These were then characterized for their physical, morphological, and mechanical features. In vitro cytotoxicity tests were conducted on ovine amniotic epithelial stem cells (oAECs), up to 7 days, to assess the effect of non-buffered and buffered PLGA by-products at different concentrations on cell viability and their stimuli on oAECs’ immunomodulatory properties. The ha-PLGA scaffolds degraded slowly as evidenced by a slight decrease in mass loss (14%) and average molecular weight (35%), with estimated degradation half-time of about 40 weeks under di-H₂O. The ultrastructure morphology of the scaffolds showed no significant fiber degradation even after 20 weeks, but alteration of fiber alignment was already evident at week 1. Moreover, mechanical properties decreased throughout the degradation times under wet as well as dry PBS conditions. The influence of acid degradation media on oAECs was dose-dependent, with a considerable effect at 7 days’ culture point. This effect was notably reduced by using buffered media. To a certain level, cells were able to compensate the generated inflammation-like microenvironment by upregulating IL-10 gene expression and favoring an anti-inflammatory rather than pro-inflammatory response. These in vitro results are essential to better understand the degradation behavior of ha-PLGA in vivo and the effect of their degradation by-products on affecting cell performance. Indeed, buffering the degradation milieu could represent a promising strategy to balance scaffold degradation. These findings give good hope with reference to the in vivo condition characterized by physiological buffering systems. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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19 pages, 3479 KiB

Open AccessArticle

A Human 3D Cardiomyocyte Risk Model to Study the Cardiotoxic Influence of X-rays and Other Noxae in Adults

by Timo Smit, Esther Schickel, Omid Azimzadeh, Christine von Toerne, Oliver Rauh, Sylvia Ritter, Marco Durante and Insa S. Schroeder

Cells 2021, 10(10), 2608; https://doi.org/10.3390/cells10102608 - 30 Sep 2021

Cited by 6 | Viewed by 2290

Abstract

The heart tissue is a potential target of various noxae contributing to the onset of cardiovascular diseases. However, underlying pathophysiological mechanisms are largely unknown. Human stem cell-derived models are promising, but a major concern is cell immaturity when estimating risks for adults. In this study, 3D aggregates of human embryonic stem cell-derived cardiomyocytes were cultivated for 300 days and characterized regarding degree of maturity, structure, and cell composition. Furthermore, effects of ionizing radiation (X-rays, 0.1–2 Gy) on matured aggregates were investigated, representing one of the noxae that are challenging to assess. Video-based functional analyses were correlated to changes in the proteome after irradiation. Cardiomyocytes reached maximum maturity after 100 days in cultivation, judged by α-actinin lengths, and displayed typical multinucleation and branching. At this time, aggregates contained all major cardiac cell types, proven by the patch-clamp technique. Matured and X-ray-irradiated aggregates revealed a subtle increase in beat rates and a more arrhythmic sequence of cellular depolarisation and repolarisation compared to non-irradiated sham controls. The proteome analysis provides first insights into signaling mechanisms contributing to cardiotoxicity. Here, we propose an in vitro model suitable to screen various noxae to target adult cardiotoxicity by preserving all the benefits of a 3D tissue culture. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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19 pages, 51257 KiB

Open AccessArticle

Ovarian Decellularized Bioscaffolds Provide an Optimal Microenvironment for Cell Growth and Differentiation In Vitro

by Georgia Pennarossa, Teresina De Iorio, Fulvio Gandolfi and Tiziana A. L. Brevini

Cells 2021, 10(8), 2126; https://doi.org/10.3390/cells10082126 - 18 Aug 2021

Cited by 16 | Viewed by 3658

Abstract

Ovarian failure is the most common cause of infertility. Although numerous strategies have been proposed, a definitive solution for recovering ovarian functions and restoring fertility is currently unavailable. One innovative alternative may be represented by the development of an “artificial ovary” that could be transplanted in patients for re-establishing reproductive activities. Here, we describe a novel approach for successful repopulation of decellularized ovarian bioscaffolds in vitro. Porcine whole ovaries were subjected to a decellularization protocol that removed the cell compartment, while maintaining the macrostructure and microstructure of the original tissue. The obtained bioscaffolds were then repopulated with porcine ovarian cells or with epigenetically erased porcine and human dermal fibroblasts. The results obtained demonstrated that the decellularized extracellular matrix (ECM)-based scaffold may constitute a suitable niche for ex vivo culture of ovarian cells. Furthermore, it was able to properly drive epigenetically erased cell differentiation, fate, and viability. Overall, the method described represents a powerful tool for the in vitro creation of a bioengineered ovary that may constitute a promising solution for hormone and fertility restoration. In addition, it allows for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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20 pages, 3308 KiB

Open AccessArticle

Generation of a Highly Biomimetic Organoid, Including Vasculature, Resembling the Native Immature Testis Tissue

by Tat-Chuan Cham, Fahar Ibtisham, Mohammad Amin Fayaz and Ali Honaramooz

Cells 2021, 10(7), 1696; https://doi.org/10.3390/cells10071696 - 5 Jul 2021

Cited by 24 | Viewed by 6903

Abstract

The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air–liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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23 pages, 4883 KiB

Open AccessArticle

Bioengineering Approaches to Improve In Vitro Performance of Prepubertal Lamb Oocytes

by Antonella Mastrorocco, Ludovica Cacopardo, Daniela Lamanna, Letizia Temerario, Giacomina Brunetti, Augusto Carluccio, Domenico Robbe and Maria Elena Dell’Aquila

Cells 2021, 10(6), 1458; https://doi.org/10.3390/cells10061458 - 10 Jun 2021

Cited by 12 | Viewed by 2919

Abstract

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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15 pages, 3080 KiB

Open AccessArticle

Metabolomic Analysis Evidences That Uterine Epithelial Cells Enhance Blastocyst Development in a Microfluidic Device

by Vanessa Mancini, Alexandra C. Schrimpe-Rutledge, Simona G. Codreanu, Stacy D. Sherrod, John A. McLean, Helen M. Picton and Virginia Pensabene

Cells 2021, 10(5), 1194; https://doi.org/10.3390/cells10051194 - 13 May 2021

Cited by 4 | Viewed by 2335

Abstract

Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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Review

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20 pages, 637 KiB

Open AccessReview

Brain and Retinal Organoids for Disease Modeling: The Importance of In Vitro Blood–Brain and Retinal Barriers Studies

by Ilenia Martinelli, Seyed Khosrow Tayebati, Daniele Tomassoni, Giulio Nittari, Proshanta Roy and Francesco Amenta

Cells 2022, 11(7), 1120; https://doi.org/10.3390/cells11071120 - 25 Mar 2022

Cited by 7 | Viewed by 4326

Abstract

Brain and retinal organoids are functional and dynamic in vitro three-dimensional (3D) structures derived from pluripotent stem cells that spontaneously organize themselves to their in vivo counterparts. Here, we review the main literature data of how these organoids have been developed through different protocols and how they have been technically analyzed. Moreover, this paper reviews recent advances in using organoids to model neurological and retinal diseases, considering their potential for translational applications but also pointing out their limitations. Since the blood–brain barrier (BBB) and blood–retinal barrier (BRB) are understood to play a fundamental role respectively in brain and eye functions, both in health and in disease, we provide an overview of the progress in the development techniques of in vitro models as reliable and predictive screening tools for BBB and BRB-penetrating compounds. Furthermore, we propose potential future directions for brain and retinal organoids, in which dedicated biobanks will represent a novel tool for neuroscience and ophthalmology research. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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17 pages, 2595 KiB

Open AccessReview

Cerebral Organoids—Challenges to Establish a Brain Prototype

by Artem V. Eremeev, Olga S. Lebedeva, Margarita E. Bogomiakova, Maria A. Lagarkova and Alexandra N. Bogomazova

Cells 2021, 10(7), 1790; https://doi.org/10.3390/cells10071790 - 15 Jul 2021

Cited by 13 | Viewed by 6755

Abstract

The new cellular models based on neural cells differentiated from induced pluripotent stem cells have greatly enhanced our understanding of human nervous system development. Highly efficient protocols for the differentiation of iPSCs into different types of neural cells have allowed the creation of 2D models of many neurodegenerative diseases and nervous system development. However, the 2D culture of neurons is an imperfect model of the 3D brain tissue architecture represented by many functionally active cell types. The development of protocols for the differentiation of iPSCs into 3D cerebral organoids made it possible to establish a cellular model closest to native human brain tissue. Cerebral organoids are equally suitable for modeling various CNS pathologies, testing pharmacologically active substances, and utilization in regenerative medicine. Meanwhile, this technology is still at the initial stage of development. Full article

(This article belongs to the Special Issue 3D Cultures, Organoids, Organ-on-Chip: New Research and Innovation Challenges in Reproduction)

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