Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection

DOI: 10.1186/s12987-022-00379-z

Authors: Leo M. Endres; Marvin Jungblut; Mustafa Divyapicigil; Markus Sauer; Christian Stigloher; Myron Christodoulides; Brandon J. Kim; Alexandra Schubert-Unkmeir

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “StemFlex medium” for “1” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should be changed once in “1” days.

Ideally, “PSC” should be obtained at the end of culture.

Step 2: Primitive streak 1

day 1 ~ 2

culture “adherent” human PSC in “DMEM/F12” for “1” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 3: endothelial 1

day 2 ~ 4

culture “adherent” human PSC in “Human endothelial cell serum-free media” for “2” days. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “EC” should express “PECAM1”, “CDH5”, “ZO-1”, “occludin”, “claudin-5” at the end of culture.

Step 4: endothelial 2

day 4 ~ 5

culture “device” human EC in “Human endothelial cell serum-free media” for “1” days. For ECM, use “collagen IV and fibronectin” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Ideally, “EC” should express “PECAM1”, “CDH5”, “ZO-1”, “occludin”, “claudin-5” at the end of culture.