For everyone conducting biological studies, this paper highlights how to think about sample size and replicates. It suggests using “SuperPlots”, which color codes data points from different experiments to better visualize the type of variability seen (i.e. random variability, experimental variability, etc…). SuperPlots: Communicating reproducibility and variability in cell biology Jim and I recommend everyone…
Introduction The basement membrane (BM) is a critical component of a healthy blood-brain barrier (BBB) model. Further, the BM breaks down in several diseases. Both pericytes and endothelial cells can contribute to basement membrane production. This post will explore a method to localize basement membrane components – collagen type IV (Col IV), fibronectin (FN), and…
Introduction While we have optimized coculture models for commercial brain endothelial cells and pericytes, we needed to do the same for iPSC-derived cocultures. In this post, I show the optimization process using IMR90-4-derived EECM-BMEC-like cells [1] and brain pericyte-like cells (BPLC) [2]. Methods The top well chip surface was coated with collagen iv (Sigma C5533), bovine fibronectin…
Introduction The µSiM BBB model is being developed at UR with the goal to understand the impact of sepsis on the brain. Here, we use an EECM-BMEC-like cell (iSPC-derived brain endothelial cells) [1] monoculture model and an EECM-BMEC-like cell and brain pericyte-like cell (BPLC; iSPC-derived brain pericytes) [2] coculture model to optimize the concentration of…
Introduction Here, I will post figure drafts for the paper that introduces the modular µSiM (m-µSiM). Figures Remaining data to obtain: Occludin staining in IMR90-4 from UniBe in µSiM. We have tried this after 2 day culture in µSiM, which worked at UR but is not working at UniBe. Current problem: Occludin is not seen in…
Introduction We are differentiating and validating EECM-BMEC (Extended Endothelial Cell Culture Method-Brain Microvascular Endothelial Cell)-like cells in µSiM devices. See this post and this post for method details and validation in traditional cell culturing platforms. Methods Attempt 1 – Overstimulated cells IMR90-4 iPSC EECM-BMEC P3 frozen vial was thawed. At P4 cells were seeded and cultured 2…
Introduction Pages regarding our in situ permeability assay development can be found in these links: Theoretical Underpinnings of Small Molecule Permeability Measurements in the µSiM (Part 1: Approach) Theoretical Underpinnings of Small Molecule Permeability Measurements in the µSiM (Part 2: Experimental Validation) Theoretical Underpinnings of Small Molecule Permeability Measurements in the µSiM (Part 3: Application to Cell…
Motivation We have discussed the optimization of our in situ permeability assay at length. This assay relies on confocal microscopy, which may not be accessible to all users of the µSiM. Therefore, we developed an alternative permeability assay in the µSiM that is an endpoint, sampling assay. The protocol for this assay can be found…
Introduction This post highlights results from my second attempt to differentiate hiPSCs into endothelial progenitor cells EECM-BMEC-like cells in our lab. For details on methodology, see Nishihara et al., FASEB, 2020 and my previous post. Methods Cells were seeded at 70,000 and 100,000 cells/well on D-3. Only the 100,000 cell/well plate was sorted. Due to media being backordered,…
Introduction Breakdown of the blood-brain barrier (BBB) is one of the earliest signs of sepsis and is linked with long-term cognitive impairment. As animal models poorly recapitulate human sepsis, alternative approaches are direly needed to understand sepsis-associated brain injury and develop strategies that protect against cognitive decline. To fill this gap, we are developing a…