Publication protocol
Cells and culture conditions: The following protocol was adapted from our previous work with minor modifications12. Cell expansion and differentiation protocols for all six cell types described here are similar to the methods in our previous work12. Primary human brain microvascular endothelial cells (Cell Systems, Kirkland, WA) were expanded in plates coated with attachment factor and were cultured under normal growth condition in complete classic medium supplemented with CultureBoostTM and attachment factor (Cell Systems). Primary human brain microvascular pericytes (HBVP; ScienCell Research Laboratories, Carlsbad, CA) were expanded in plates coated with 15 μg/ml Poly-L Lysine (ScienCell Research Laboratories) and were cultured under normal growth conditions in pericyte medium (ScienCell Research Laboratories) supplemented with 2% FBS, pericyte growth supplement and penicillin-streptomycin. Human astrocytes (HA) (ScienCell Research Laboratories) were propagated in plates coated with 0.2 mg/mL Matrigel (Corning) and were cultured under normal growth condition in astrocyte medium (ScienCell Research Laboratories) containing 2% FBS, astrocyte growth supplement, and penicillin-streptomycin. Human iPSC-derived oligodendrocyte progenitor cells (HO; Tempo Bioscience Inc., San Francisco, CA) were propagated in plates coated with 0.2 mg/mL Matrigel (Corning) and were cultured under normal growth conditions in (a) propagation media consisting of DMEM/F12 with HEPES, L- glutamine (2 mM, Life Technologies), non-Essential amino acids (1X Life Technologies), StemPro neural supplement (Invitrogen), PDGF-AA (10 ng/mL, Peprotech), PDGF-AB (10 ng/mL, Peprotech), NT3 (10 ng/mL, Peprotech), Biotin (100 ng/mL, Sigma Aldrich), and cAMP (5 μM/mL Sigma Aldrich) and were then cultured in (b) differentiation media (for 72 hrs prior to organoid formation) consisting of 50:50 DMEM/F12:neuralbasal (Life Technologies), non-essential amino acids (1X Life Technologies), 1x B27 (Life Technologies), L- glutamine (2 mM, Life Technologies), Biotin (100 ng/ml, Sigma Aldrich), PDGF-AA (5 ng/mL, Peprotech), BDNF (10 ng/ml, Peprotech), ascorbic acid (20 μg/mL, Sigma Aldrich), cAMP (1 μM/ml Sigma Aldrich), T3 (200 ng/ml, (Sigma Aldrich). Human iPSC- derived microglia (HM; Tempo Bioscience Inc., San Francisco, CA) were propagated in plates coated with 0.2 mg/ml Matrigel (Corning) and were cultured under normal growth conditions in DMEM/F-12 (Life Technologies), N2 supplement (1×, Life Technologies), essential amino acids (0.5×, Life Technologies) L-glutamine 2 mM, LifeTech), GM-CSF (100 ng/mL, Peprotech), IL-34 (50 ng/mL, Peprotech). Human iPSC- derived neural stem cells (HN) (Axol Biosciences Ltd., Cambridge, UK) were plated on plates coated with SureBond (Axol Biosciences) and cultured under normal growth conditions in 50% neural plating-XF medium (Axol Biosciences) and 50% neural expansion-XF medium (Axol Biosciences) supplemented with recombinant human FGF2 (20 ng/mL, Axol Biosciences) and recombinant human EGF (20 ng/mL, Axol Biosciences). After 48 hours, the media was then replaced with 100% neural expansion-XF medium (Axol Biosciences) supplemented with recombinant human FGF2 (20 ng/mL, Axol Biosciences) and recombinant human EGF (20 ng/mL, Axol Biosciences). Organoid culture: The following protocol was adapted from our previous work with minor modifications for some experiements12. HBMEC and HBVP were harvested using TrypLE select enzyme (1X Life Technologies), HA, HM and HO were harvested from culture plates with accutase (Life Technologies) and HN were harvested with unlock-XF (Axol Bioscience). The organoids contained 30% HBMEC, 15% HBVP, 15% HA, 5% HM, 15% HO, and 20% HN with approximately 2000 cells per organoid, except for organoids in Fig. 7A,C and andDcontainedDcontained 4000 cells each. Organoids containing HA, HM, HO and HN were allowed to form in 50% astrocyte medium without astrocyte growth supplements and 50% neural maintenance-XF medium under normal growth conditions for 48 hrs using the hanging drop culture method in hanging drop culture plates (InSphero AG, Schlieren, Switzerland). The medium was mixed with heat inactivated FBS (Thermo Fisher) and 10 ng/μL rat tail collagen I (Corning). HBMEC and HBVP suspended in 50% Astrocyte media and 50% complete classic media were subsequently added to the neural-glial organoid thereby allowing the two cell types to coat the surface of the neuro-glia organoid. The organoids were cultured under normal growth conditions in 60% neural maintenance-XF medium, 20% astrocyte medium and 20% complete classic medium (Organoid Media). The organoids were then allowed to mature further for 48 hrs and were dropped into a 96 well plate for long term use. The organoids used in all subsequent studies were between 6–12 days in vitro.
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