Culture of iPSC-BMECs at RIT

Introduction

The capability of the m-μSiM for establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and bioscience laboratories has been shown previously [1]. To further demonstrate interlaboratory agreement and successful dissemination of components and protocols, m-μSiM was used at RIT (Abhyankar lab) to establish an hiPSC-BMECs monolayer.

Method

All reagents and steps were used based on the provided protocol by Engelhardt lab [2]. uSiM devices with NPN membranes were treated with the coating solution (Collagen IV: 4 units, Fibronectin: 1 unit, Water: 5 units) for 3 hrs in the incubator. iPSC-BMECs were split using Accutase (3 min) and seeded on each device at a density of 50,000 cells/cm2. The media was changed after 2 hrs of cell seeding. Immunostaining was conducted based on the protocol provided by McGrath lab.

Results

• We have been able to selectively passage iPSC-BMECs from EPCs provided by McGrath lab.
• We have been able to maintain the culture of iPSC-BMECs on uSiM for 6 days.
• We have been able to visualize Claudin 5 and ZO-1 after 4 days of culture, but not occludin (Fig. 1)

Reference

[1] 1. Molly C. McCloskey, Pelin Kasap, S. Danial Ahmad, Shiuan‐Haur Su, Kaihua Chen, Mehran Mansouri, Natalie Ramesh et al. “The Modular μSiM: a Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro”, Advanced healthcare materials (2022)

[2] Nishihara, Hideaki, et al. “Differentiation of human pluripotent stem cells to brain microvascular endothelial cell-like cells suitable to study immune cell interactions.” STAR protocols 2.2 (2021): 100563.