bims-orenst 2021-08-08 papers

Issue of 2021–08–08
seven papers selected by
Joram Mooiweer, University of Groningen

Lab Chip. 2021 Aug 04.

Simulating drug concentrations in PDMS microfluidic organ chips.

Jennifer Grant, Alican Özkan, Crystal Oh, Gautam Mahajan, Rachelle Prantil-Baun, Donald E Ingber.

Microfluidic organ-on-a-chip (Organ Chip) cell culture devices are often fabricated using polydimethylsiloxane (PDMS) because it is biocompatible, transparent, elastomeric, and oxygen permeable; however, hydrophobic small molecules can absorb to PDMS, which makes it challenging to predict drug responses. Here, we describe a combined simulation and experimental approach to predict the spatial and temporal concentration profile of a drug under continuous dosing in a PDMS Organ Chip containing two parallel channels separated by a porous membrane that is lined with cultured cells, without prior knowledge of its log P value. First, a three-dimensional finite element model of drug loss into the chip was developed that incorporates absorption, adsorption, convection, and diffusion, which simulates changes in drug levels over time and space as a function of potential PDMS diffusion coefficients and log P values. By then experimentally measuring the diffusivity of the compound in PDMS and determining its partition coefficient through mass spectrometric analysis of the drug concentration in the channel outflow, it is possible to estimate the effective log P range of the compound. The diffusion and partition coefficients were experimentally derived for the antimalarial drug and potential SARS-CoV-2 therapeutic, amodiaquine, and incorporated into the model to quantitatively estimate the drug-specific concentration profile over time measured in human lung airway chips lined with bronchial epithelium interfaced with pulmonary microvascular endothelium. The same strategy can be applied to any device geometry, surface treatment, or in vitro microfluidic model to simulate the spatial and temporal gradient of a drug in 3D without prior knowledge of the partition coefficient or the rate of diffusion in PDMS. Thus, this approach may expand the use of PDMS Organ Chip devices for various forms of drug testing.

DOI: https://doi.org/10.1039/d1lc00348h

Hum Reprod. 2021 Aug 07. pii: deab186. [Epub ahead of print]

Three-dimensional microengineered vascularised endometrium-on-a-chip.

Jungho Ahn, Min-Ji Yoon, Seon-Hwa Hong, Hwijae Cha, Danbi Lee, Hwa Seon Koo, Ji-Eun Ko, Jungseub Lee, Soojung Oh, Noo Li Jeon, Youn-Jung Kang.

STUDY QUESTION: Can we reconstitute physiologically relevant 3-dimensional (3D) microengineered endometrium in-vitro model?
SUMMARY ANSWER: Our representative microengineered vascularised endometrium on-a-chip closely recapitulates the endometrial microenvironment that consists of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D extracellular matrix in a spatiotemporal manner.
WHAT IS KNOWN ALREADY: Organ-on-a-chip, a multi-channel 3D microfluidic cell culture system, is widely used to investigate physiologically relevant responses of organ systems.
STUDY DESIGN, SIZE, DURATION: The device consists of five microchannels that are arrayed in parallel and partitioned by array of micropost. Two central channels are for 3D culture and morphogenesis of stromal fibroblast and endothelial cells. In addition, the outermost channel is for the culture of additional endometrial stromal fibroblasts that secrete biochemical cues to induce directional pro-angiogenic responses of endothelial cells. To seed endometrial epithelial cells, on Day 8, Ishikawa cells were introduced to one of the two medium channels to adhere on the gel surface. After that, the microengineered endometrium was cultured for an additional 5-6 days (total ∼ 14 days) for the purpose of each experiment.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Microfluidic 3D cultures were maintained in endothelial growth Medium 2 with or without oestradiol and progesterone. Some cultures additionally received exogenous pro-angiogenic factors. For the three distinct layers of microengineered endometrium-on-a-chip, the epithelium, stroma and blood vessel characteristics and drug response of each distinct layer in the microfluidic model were assessed morphologically and biochemically. The quantitative measurement of endometrial drug delivery was evaluated by the permeability coefficients.
MAIN RESULTS AND THE ROLE OF CHANCE: We established microengineered vascularised endometrium-on-chip, which consists of three distinct layers: epithelium, stroma and blood vessels. Our endometrium model faithfully recapitulates in-vivo endometrial vasculo-angiogenesis and hormonal responses displaying key features of the proliferative and secretory phases of the menstrual cycle. Furthermore, the effect of the emergency contraception drug levonorgestrel was evaluated in our model demonstrating increased endometrial permeability and blood vessel regression in a dose-dependent manner. We finally provided a proof of concept of the multi-layered endometrium model for embryo implantation, which aids a better understanding of the molecular and cellular mechanisms underlying this process.
LARGE SCALE DATA: N/A.
LIMITATIONS, REASONS FOR CAUTION: This report is largely an in-vitro study and it would be beneficial to validate our findings using human primary endometrial cells.
WIDER IMPLICATIONS OF THE FINDINGS: Our 3D microengineered vascularised endometrium-on-a-chip provides a new in-vitro approach to drug screening and drug discovery by mimicking the complicated behaviours of human endometrium. Thus, we suggest our model as a tool for addressing critical challenges and unsolved problems in female diseases, such as endometriosis, uterine cancer and female infertility, in a personalised manner.
STUDY FUNDING/COMPETING INTEREST(S): This work is supported by funding from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) to Y.J.K. (No. 2018R1C1B6003), to J.A. (No. 2020R1I1A1A01074136) and to H.S.K. (No. 2020R1C1C100787212). The authors report no conflicts of interest.

Keywords: 3D culture; drug screening; endometrial angiogenesis; endometrium; microfluidic

DOI: https://doi.org/10.1093/humrep/deab186

Int J Mol Sci. 2021 Jul 30. pii: 8234. [Epub ahead of print]22(15):

A Microfluidic 3D Endothelium-on-a-Chip Model to Study Transendothelial Migration of T Cells in Health and Disease.

Luuk de Haan, Johnny Suijker, Ruthger van Roey, Nina Berges, Elissaveta Petrova, Karla Queiroz, Wouter Strijker, Thomas Olivier, Oliver Poeschke, Sakshi Garg, Lenie J van den Broek.

The recruitment of T cells is a crucial component in the inflammatory cascade of the body. The process involves the transport of T cells through the vascular system and their stable arrest to vessel walls at the site of inflammation, followed by extravasation and subsequent infiltration into tissue. Here, we describe an assay to study 3D T cell dynamics under flow in real time using a high-throughput, artificial membrane-free microfluidic platform that allows unimpeded extravasation of T cells. We show that primary human T cells adhere to endothelial vessel walls upon perfusion of microvessels and can be stimulated to undergo transendothelial migration (TEM) by TNFα-mediated vascular inflammation and the presence of CXCL12 gradients or ECM-embedded melanoma cells. Notably, migratory behavior was found to differ depending on T cell activation states. The assay is unique in its comprehensiveness for modelling T cell trafficking, arrest, extravasation and migration, all in one system, combined with its throughput, quality of imaging and ease of use. We envision routine use of this assay to study immunological processes and expect it to spur research in the fields of immunological disorders, immuno-oncology and the development of novel immunotherapeutics.

Keywords: T cell; extravasation; high-throughput; immuno-oncology; in vitro; inflammation; microfluidic; organ-on-a-chip; transendothelial migration

DOI: https://doi.org/10.3390/ijms22158234

Nat Commun. 2021 Aug 05. 12(1): 4730

Microfluidic device with brain extracellular matrix promotes structural and functional maturation of human brain organoids.

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.

DOI: https://doi.org/10.1038/s41467-021-24775-5

Biomaterials. 2021 Jul 20. pii: S0142-9612(21)00331-8. [Epub ahead of print]276 120975

The driving role of the Cdk5/Tln1/FAKS732 axis in cancer cell extravasation dissected by human vascularized microfluidic models.

Mara Gilardi, Simone Bersini, Silvia Valtorta, Marco Proietto, Martina Crippa, Alexandra Boussommier-Calleja, Myriam Labelle, Rosa Maria Moresco, Marco Vanoni, Roger D Kamm, Matteo Moretti.

BACKGROUND: Understanding the molecular mechanisms of metastatic dissemination, the leading cause of death in cancer patients, is required to develop novel, effective therapies. Extravasation, an essential rate-limiting process in the metastatic cascade, includes three tightly coordinated steps: cancer cell adhesion to the endothelium, trans-endothelial migration, and early invasion into the secondary site. Focal adhesion proteins, including Tln1 and FAK, regulate the cytoskeleton dynamics: dysregulation of these proteins is often associated with metastatic progression and poor prognosis.
METHODS: Here, we studied the previously unexplored role of these targets in each extravasation step using engineered 3D in vitro models, which recapitulate the physiological vascular niche experienced by cancer cells during hematogenous metastasis.
RESULTS: Human breast cancer and fibrosarcoma cell lines respond to Cdk5/Tln1/FAK axis perturbation, impairing their metastatic potential. Vascular breaching requires actin polymerization-dependent invadopodia formation. Invadopodia generation requires the structural function of FAK and Tln1 rather than their activation through phosphorylation. Our data support that the inhibition of FAKS732 phosphorylation delocalizes ERK from the nucleus, decreasing ERK phosphorylated form. These findings indicate the critical role of these proteins in driving trans-endothelial migration. In fact, both knock-down experiments and chemical inhibition of FAK dramatically reduces lung colonization in vivo and TEM in microfluidic setting. Altogether, these data indicate that engineered 3D in vitro models coupled to in vivo models, genetic, biochemical, and imaging tools represent a powerful weapon to increase our understanding of metastatic progression.
CONCLUSIONS: These findings point to the need for further analyses of previously overlooked phosphorylation sites of FAK, such as the serine 732, and foster the development of new effective antimetastatic treatments targeting late events of the metastatic cascade.

Keywords: Breast cancer; Cdk5; Extravasation; FAK; Fibrosarcoma; Focal adhesion; Metastasis; Microfluidic; Tln1; Vascular niche

DOI: https://doi.org/10.1016/j.biomaterials.2021.120975

Sci Rep. 2021 Aug 04. 11(1): 15840

Co-culture model of B-cell acute lymphoblastic leukemia recapitulates a transcription signature of chemotherapy-refractory minimal residual disease.

Stephanie L Rellick, Gangqing Hu, Debra Piktel, Karen H Martin, Werner J Geldenhuys, Rajesh R Nair, Laura F Gibson.

B-cell acute lymphoblastic leukemia (ALL) is characterized by accumulation of immature hematopoietic cells in the bone marrow, a well-established sanctuary site for leukemic cell survival during treatment. While standard of care treatment results in remission in most patients, a small population of patients will relapse, due to the presence of minimal residual disease (MRD) consisting of dormant, chemotherapy-resistant tumor cells. To interrogate this clinically relevant population of treatment refractory cells, we developed an in vitro cell model in which human ALL cells are grown in co-culture with human derived bone marrow stromal cells or osteoblasts. Within this co-culture, tumor cells are found in suspension, lightly attached to the top of the adherent cells, or buried under the adherent cells in a population that is phase dim (PD) by light microscopy. PD cells are dormant and chemotherapy-resistant, consistent with the population of cells that underlies MRD. In the current study, we characterized the transcriptional signature of PD cells by RNA-Seq, and these data were compared to a published expression data set derived from human MRD B-cell ALL patients. Our comparative analyses revealed that the PD cell population is markedly similar to the MRD expression patterns from the primary cells isolated from patients. We further identified genes and key signaling pathways that are common between the PD tumor cells from co-culture and patient derived MRD cells as potential therapeutic targets for future studies.

DOI: https://doi.org/10.1038/s41598-021-95039-x

J Neurosci Methods. 2021 Jul 31. pii: S0165-0270(21)00237-5. [Epub ahead of print] 109302

Stretchable microchannel-on-a-chip: A simple model for evaluating the effects of uniaxial strain on neuronal injury.

Sam Parittotokkaporn, Anusha Dravid, Brad J Raos, Samuel Rosset, Darren Svirskis, Simon J O'Carroll.

BACKGROUND: Axonal injury is a major component of traumatic spinal cord injury (SCI), associated with rapid deformation of spinal tissue and axonal projections. In vitro models enable us to examine these effects and screen potential therapies in a controlled, reproducible manner.
NEW METHOD: A customised, stretchable microchannel system was developed using polydimethylsiloxane microchannels. Cortical and spinal embryonic rat neurons were cultured within the microchannel structures, allowing a uniaxial strain to be applied to isolated axonal processes. Global strains of up to 52% were applied to the stretchable microchannel-on-a-chip platform leading to local strains of up to 12% being experienced by axons isolated in the microchannels.
RESULTS: Individual axons exposed to local strains between 3.2% to 8.7% developed beading within 30-minutes of injury. At higher local strains of 9.8% and 12% individual axons ruptured within 30-minutes of injury. Axon bundles, or fascicles, were more resistant to rupture at each strain level, compared to individual axons. At lower local strain of 3.2%, axon bundles inside microchannels and neuronal cells near entrances of them progressively swelled and degenerated over a period of 7 days after injury.
COMPARISON WITH EXISTING METHOD(S): This method is simple, reliable and reproducible with good control and measurement of injury tolerance and morphological deformations using standard laboratory equipment. By measuring local strains, we observed that axonal injuries occur at a lower strain magnitude and a lower strain rate than previous methods reporting global strains, which may not accurately reflect the true strain.
CONCLUSIONS: We describe a novel stretchable microchannel-on-a-chip platform to study the effect of varying local strain on morphological characteristics of neuronal injury.

Keywords: Axonal injury; Axotomy; Microchannel; Neuronal injury; Uniaxial strain

DOI: https://doi.org/10.1016/j.jneumeth.2021.109302