Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.Several clinical reports have described gastrointestinal symptoms for COVID-19, though whether the virus can replicate within the stomach remains unclear. Here the authors generate gastric organoids from human biopsies and show that the virus can efficiently infect gastric epithelium, suggesting that the stomach might have an active role in fecal-oral transmission

Microtechnology-aided differentiation of human pluripotent stem cells into hepatocyte-like cells

There is a growing interest by scientific community, together with pharmaceutical companies and clinical researcher on finding valid alternatives to standard models in the development of new therapeutic strategies and robust drug screening processes. Research still relies on the use of immortalized cell lines, primary cell extracted from living organs and animal models. These methodologies are valid and still mainstream for general purposes, but most of the times present evident limits. Cell lines and primary cells often fail to reproduce the exact characteristics of the tissue found in vivo, because they lose some of the features and functionalities when grown in culture. On the other hand, animal models, even if necessary for the study of specific diseases and new compound testing, are expensive and time-consuming, and often show poor predictive capacity and scarce reproducibility of the human condition. Hence, human pluripotent stem cells could represent a valid alternative to the existing models, thank to their capacity to be expanded indefinitely and differentiate into almost all cell types found in vivo. In the recent years, there has been a growing attention on engineered tissues differentiate from pluripotent cells. They have the capacity to transform the way to study human pathophysiology and physiology in vitro. Nevertheless, there are still major problems in the process of differentiating human embryonic and induced pluripotent stem cell in vitro. This is because of the difficulty to specifically direct cell fate to a particular cell type in a robust way, and for the poor reproduction of the physiological conditions under which these processes take place. In this direction, new microtechnologies could help overcome these major limitations, because they allow working on microscales, in a way that cannot be reproduced by standard culture conditions. In this context, the aim of this PhD thesis is to efficiently differentiate human pluripotent stem cells in order to obtain functional relevant cell types, such as cardiomyocytes and hepatocytes. The strategy applied for the obtainment of these human in vitro models rely on the application of microscale technologies for reproducing in vitro the main physiological cues, which guide differentiation and allow functional development. In particular, three-dimensional microwell were used for modulating endogenous factor accumulation on differentiating human embryoid bodies, to screen for selective germ layer commitment guided by a differential microenvironment around the differentiating cells. Furthermore, a mechanical modulation of the pluripotent nuclei mechanical properties was imposed with the use of microstructured substrate, for the study of the peculiar capacity of nuclear cell deformation, and its effect in pluripotency and early differentiation. Moreover, microfluidic technologies were used to selectively modulate cell soluble microenvironment, in order to optimize pluripotency maintenance, early germ layer commitment, and functional differentiation into cardiomyocytes and hepatocytes. A high percentage of spontaneously beating cardiomyocytes on chip was obtained, showing proper functional response to calcium stimuli. On the other hand, microfluidic technology allowed to obtain a higher percentage of hepatocytes compared to standard culture conditions. These cells showed proper phenotypic and functional characteristics, which were also analyzed in a more physiological condition under a defined oxygen gradient, mimicking in vivo physiological conditions. These specific cell types generated on chip from human pluripotent stem cells through a multi-stage approach show specific functional differentiation, which opens a new perspective for multi-parametric and large scale human tissue-based screening assays.Vi è un crescente interesse fra la comunità scientifica internazionale, le compagnie farmaceutiche e la ricerca clinica nel trovare delle valide alternative ai modelli standard nello sviluppo di nuove strategie terapeutiche e di processi di scoperta di nuovi farmaci. I ricercatori si basano tuttora sull’utilizzo di linee cellulari immortalizzate, cellule primarie estratte da organi e su modelli animali. Queste metodiche sono valide e largamente utilizzate per scopi generali, ma il più delle volte presentano dei limiti evidenti. Le linee cellulari e cellule primarie spesso non riproducono fedelmente le esatte caratteristiche dei tessuti in vivo, poiché perdono alcune caratteristiche fisiche e funzionali quando sono coltivate in vitro. D’altra parte, i modelli animali sono ancora necessari per lo studio di patologie specifiche e nel test di nuovi composti, ma risultano essere dispendiosi in termini di tempo e denaro e spesso mostrano una scarsa capacità predittiva nei confronti degli effetti sull’uomo. Quindi, le cellule staminali pluripotenti umane rappresentano una valida alternativa ai modelli esistenti, grazie alle loro capacità di essere espanse in vitro indefinitamente e di poter differenziare in tutti i tipi cellulari derivanti dai tre foglietti germinali. Negli ultimi anni l’attenzione si è concentrata sui tessuti ingegnerizzati, differenziati a partire da cellule pluripotenti. Questi hanno la possibilità di trasformare radicalmente il modo in cui studiamo in vitro la fisiologia e la patofisiologia umana. Ciò non di meno, vi sono ancora problemi nei processi di differenziamento di cellule staminali embrionali e pluripotenti indotte umane in vitro. Questo perché si riscontra difficoltà nel dirigere specificamente il destino cellulare verso un determinato tipo cellulare in modo robusto, e per la scarsa riproducibilità delle condizioni fisiologiche in cui questi processi hanno normalmente luogo in vivo. In questo ambito, le nuove micro-tecnologie possono dare un aiuto nell’oltrepassare queste limitazioni, poiché permettono di lavorare in micro-scala in maniera difficilmente riproducibile in condizioni di coltura standard. In questo contesto, lo scopo di questa tesi di dottorato è quello di differenziare efficacemente cellule staminali pluripotenti umane per riuscire ad ottenere rilevanti tipi cellulari, quali cardiomiociti ed epatociti. La strategia applicata per l’ottenimento di questi modelli umani in vitro si basa sull’applicazione di tecnologie in micro-scala per permettere la riproduzione in vitro delle nicchie fisiologiche, che guidano il differenziamento e permettono lo sviluppo funzionale. In particolare sono stati utilizzati dei micro-pozzetti tridimensionali per modulare l’accumulo di fattori endogeni in corpi embrioidi umani in differenziamento, per studiare lo sviluppo dei tre foglietti germinali guidato da diversi microambienti cellulari. È stata poi imposta una modulazione delle proprietà meccaniche dei nuclei di cellule pluripotenti tramite utilizzo di substrati micro-strutturati, per lo studio della capacità peculiare di deformazione nucleare di tali cellule e si è valutato l’effetto sulla pluripotenza e il differenziamento precoce. Inoltre sono state utilizzate tecnologie micro-fluidiche per modulare selettivamente il microambiente cellulare solubile, in modo da ottimizzare il mantenimento della pluripotenza in chip micro-fluidici, nonché lo sviluppo cellulare precoce e il differenziamento funzionale in cardiomiociti ed epatociti. È stata ottenuta un’alta percentuale di cardiomiociti contrattili nei chip che mostravano risposte funzionali corrette a stimoli di calcio. La tecnologia micro-fluidica ha permesso poi di ottenere un’alta percentuale di epatociti in chip rispetto alle condizioni di coltura standard. Queste cellule mostravano caratteristiche fenotipiche e funzionali corrette, che sono state poi analizzate in condizioni più fisiologiche sotto un gradiente stabile di ossigeno, mimando le condizioni in vivo. Questi tipi cellulari specifici, generati in chip da cellule staminali pluripotenti umane tramite un approccio multi-stadio, mostrano un differenziamento funzionale specifico, che apre a nuove prospettive per test multi-parametrici su larga scala basati su tessuti funzionali umani

Differentiation Of Human Pluripotent Stem Cells Into Hepatic Cells And Development Of A Liver Tissue On A Chip

none4The therapeutic potential of human pluripotent stem (hPS) cells is threatened by the difficulty to homogeneously direct cell differentiation into specific lineages. Aims: a) to efficiently derive mature hepatic cells from hPS cells; b) to integrate the specific lineages into a microfluidic platform to obtain a functional liver tissue on a chip. Methods: Human embryonic stem cells (cell line HES2, National Stem Cell Bank, Madison WI) and induced hPS cells (cell line ADHF#1, iCEMS, Kyoto University) were grown on mouse embryonic fibroblasts (Chemicon, Temecula, CA) and were differentiated on matrigel. Then we developed a multi-stage microfluidic technology to derive mature cells from stem cells. Obtained cells have been characterized both with hepatic markers (alpha-fetoprotein, cytokeratins 18, 19, albumin, CYP3A) and functional tests (glycogen storage, indocyanine green uptake, albumin secretion). Results: We efficiently differentiated both human embryonic and induced pluripotent stem cells. Two hepatic lineages were obtained: hepatocyte- and cholangiocyte-like cells showing high CYP3A expression, indocyanine uptake, glycogen storage and albumin secretion over a 14-day period. This technology allowed to accurately control hPS cells expansion and fate toward early endoderm commitment, hepatic development and functional maturation on a chip. Compared to conventional culture, microfluidic platform allowed shortening of the time required for differentiation and enhanced functional activity. The proportion between hepatocyte- and cholangiocyte-like cells was 3:1. In particular, we obtained 75% of cells with glycogen storage capacity, whereas the number of CYP3A-positive cells resulted in a 59% of the total, with a 20% increase compared to the standard hepatocytes differentiation. Albumin production was about 40% higher than standard conditions. Conclusions: The engineerization of pluripotent cell differentiation into hepatic lineages will allow us to further understand the mechanisms involved in tissue development. Moreover, mature hepatic cells fully integrated on a chip could be directly used for temporal-defined toxicological assays and drug screening.noneG.G. Giobbe; F. Michielin; N. Elvassore; A. FloreaniGiobbe, GIOVANNI GIUSEPPE; Michielin, Federica; Elvassore, Nicola; Floreani, Annaros

Controlled cardiac differentiation of human embryonic stem cell-derived embryoid bodies in scalable bioreactors

none7noneZAGALLO M; LUNI C; SERENA E; CIMETTA E; ZATTI S.; GIOBBE G; ELVASSORE NZagallo, Monica; Luni, Camilla; Serena, Elena; Cimetta, Elisa; Zatti, S.; Giobbe, GIOVANNI GIUSEPPE; Elvassore, Nicol