bims-orenst 2021-06-06 papers

Issue of 2021–06–06
five papers selected by
Joram Mooiweer, University of Groningen

Biomed Microdevices. 2021 May 31. 23(2): 30

Highly parallelized human embryonic stem cell differentiation to cardiac mesoderm in nanoliter chambers on a microfluidic chip.

Anke R Vollertsen, Simone A Ten Den, Verena Schwach, Albert van den Berg, Robert Passier, Andries D van der Meer, Mathieu Odijk.

Human stem cell-derived cells and tissues hold considerable potential for applications in regenerative medicine, disease modeling and drug discovery. The generation, culture and differentiation of stem cells in low-volume, automated and parallelized microfluidic chips hold great promise to accelerate the research in this domain. Here, we show that we can differentiate human embryonic stem cells (hESCs) to early cardiac mesodermal cells in microfluidic chambers that have a volume of only 30 nanoliters, using discontinuous medium perfusion. 64 of these chambers were parallelized on a chip which contained integrated valves to spatiotemporally isolate the chambers and automate cell culture medium exchanges. To confirm cell pluripotency, we tracked hESC proliferation and immunostained the cells for pluripotency markers SOX2 and OCT3/4. During differentiation, we investigated the effect of different medium perfusion frequencies on cell reorganization and the expression of the early cardiac mesoderm reporter MESP1mCherry by live-cell imaging. Our study demonstrates that microfluidic technology can be used to automatically culture, differentiate and study hESC in very low-volume culture chambers even without continuous medium perfusion. This result is an important step towards further automation and parallelization in stem cell technology.

Keywords: Cardiac mesoderm; Discontinuous perfusion; Human pluripotent stem cells; Microfluidic large-scale integration; Parallelization

DOI: https://doi.org/10.1007/s10544-021-00556-1

Int J Mol Sci. 2021 May 19. pii: 5361. [Epub ahead of print]22(10):

A 3D Renal Proximal Tubule on Chip Model Phenocopies Lowe Syndrome and Dent II Disease Tubulopathy.

Sindhu Naik, Andrew R Wood, Maté Ongenaert, Paniz Saidiyan, Edo D Elstak, Henriëtte L Lanz, Jan Stallen, Richard Janssen, Elizabeth Smythe, Kai S Erdmann.

Lowe syndrome and Dent II disease are X-linked monogenetic diseases characterised by a renal reabsorption defect in the proximal tubules and caused by mutations in the OCRL gene, which codes for an inositol-5-phosphatase. The life expectancy of patients suffering from Lowe syndrome is largely reduced because of the development of chronic kidney disease and related complications. There is a need for physiological human in vitro models for Lowe syndrome/Dent II disease to study the underpinning disease mechanisms and to identify and characterise potential drugs and drug targets. Here, we describe a proximal tubule organ on chip model combining a 3D tubule architecture with fluid flow shear stress that phenocopies hallmarks of Lowe syndrome/Dent II disease. We demonstrate the high suitability of our in vitro model for drug target validation. Furthermore, using this model, we demonstrate that proximal tubule cells lacking OCRL expression upregulate markers typical for epithelial-mesenchymal transition (EMT), including the transcription factor SNAI2/Slug, and show increased collagen expression and deposition, which potentially contributes to interstitial fibrosis and disease progression as observed in Lowe syndrome and Dent II disease.

Keywords: Lowe syndrome; OCRL; disease modeling; fibrosis; microfluidic; organ-on-a-chip; proximal tubule-on-a-chip

DOI: https://doi.org/10.3390/ijms22105361

Cancers (Basel). 2021 May 18. pii: 2461. [Epub ahead of print]13(10):

A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments.

Valentina Palacio-Castañeda, Simon Dumas, Philipp Albrecht, Thijmen J Wijgers, Stéphanie Descroix, Wouter P R Verdurmen.

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s-1 and 2 µm2 ·s-1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

Keywords: DARPin; protein diffusion; spheroid; tumor targeting; tumor-on-a-chip

DOI: https://doi.org/10.3390/cancers13102461

Cells. 2021 May 20. pii: 1268. [Epub ahead of print]10(5):

Establishing a 3D In Vitro Hepatic Model Mimicking Physiologically Relevant to In Vivo State.

Hyun Kyoung Kang, Madina Sarsenova, Da-Hyun Kim, Min Soo Kim, Jin Young Lee, Eun-Ah Sung, Myung Geun Kook, Nam Gyo Kim, Soon Won Choi, Vyacheslav Ogay, Kyung-Sun Kang.

Three-dimensional (3D) bioprinting is a promising technology to establish a 3D in vitro hepatic model that holds great potential in toxicological evaluation. However, in current hepatic models, the central area suffers from hypoxic conditions, resulting in slow and weak metabolism of drugs and toxins. It remains challenging to predict accurate drug effects in current bioprinted hepatic models. Here, we constructed a hexagonal bioprinted hepatic construct and incorporated a spinning condition with continuous media stimuli. Under spinning conditions, HepG2 cells in the bioprinted hepatic construct exhibited enhanced proliferation capacity and functionality compared to those under static conditions. Additionally, the number of spheroids that play a role in boosting drug-induced signals and responses increased in the bioprinted hepatic constructs cultured under spinning conditions. Moreover, HepG2 cells under spinning conditions exhibited intensive TGFβ-induced epithelial-to-mesenchymal transition (EMT) and increased susceptibility to acetaminophen (APAP)-induced hepatotoxicity as well as hepatotoxicity prevention by administration of N-acetylcysteine (NAC). Taken together, the results of our study demonstrate that the spinning condition employed during the generation of bioprinted hepatic constructs enables the recapitulation of liver injury and repair phenomena in particular. This simple but effective culture strategy facilitates bioprinted hepatic constructs to improve in vitro modeling for drug effect evaluation.

Keywords: 3D bioprinting; dynamic environment; in vitro model; liver

DOI: https://doi.org/10.3390/cells10051268

J Am Soc Nephrol. 2021 Jun 02. pii: ASN.2021020231. [Epub ahead of print]

REST and Stress Resistance in the Aging Kidney.

Sato Magassa, Liviu Aron, Clément Hoguin, Bruce Yankner, Pierre Isnard, Fabiola Terzi, Christophe Legendre, Guillaume Canaud.

BACKGROUND: Chronic kidney disease is associated with the loss of functional nephrons, leading to increased mechanical and metabolic stress in the remaining cells, particularly for cells constituting the filtration barrier, such as podocytes. The failure of podocytes to mount an adequate stress response can lead to further nephron loss and disease progression. However, the mechanisms that regulate this degenerative process in the kidney are unknown.
METHODS: We combined in vitro, in vivo, and organ-on-chip approaches to identify the RE1-silencing transcription factor (REST), a repressor of neuronal genes during embryonic development, as a central regulator of podocyte adaptation to injury and aging.
RESULTS: Mice with a specific deletion of REST in podocytes exhibit albuminuria, podocyte apoptosis, and glomerulosclerosis during aging, and exhibit increased vulnerability to renal injury. This phenotype is mediated, in part, by effects of REST on the podocyte cytoskeleton that promote resistance to mechanical stressors and augment podocyte survival. Finally, REST expression is upregulated in human podocytes during aging, consistent with a conserved mechanism of stress resistance.
CONCLUSIONS: These results suggest that REST protects the kidney from injury and degeneration during aging, with potentially important therapeutic implications.

DOI: https://doi.org/10.1681/ASN.2021020231