Development and Maintenance of Serum-Free Embryoid Bodies

Samenvatting

Source: Phillips, A. W., et al. Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays. J. Vis. Exp. (2017).

The video describes the procedure for developing serum-free embryoid bodies (SFEBs) from neuronal progenitor cells using various serum-free differentiation mediums or DM. DM-2 facilitates neurons' maturation and arrangement to form SFEBs, while DM-3 is used for further growth maintenance.

Protocol

1. Serum-Free Embryoid Body (SFEB) Induction (Figure 1)

1. Place 40 µm cell culture inserts into 6-well plates and add 1 mL differentiation medium 1 (DM1) (Table of Materials) at least 1 h before adding aggregates.
2. Transfer the neural progenitor cell aggregates with 20 µL medium using a 200 µL wide mouth tip onto 40 µm cell culture inserts and change the medium to DM2 (Table of Materials).
   NOTE: Cell culture inserts are specialized inserts that sit slightly off the bottom of the well and consist of a polytetrafluoroethylene membrane suspended across a plastic frame. This m....

Resultaten

Figure 1: Timeline of Medium Changes and Phases during SFEB Growth and Expansion.

After SFEBs have been transferred to inserts, they undergo two medium changes at 14 and 28 days in vitro (DIV). They are.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
SFEB Neuronal Differentiation Cell culture Media. Reagents. Components
STEMdiff Neural Induction Medium (hiPSC Media)	STEMCELL Technologies	0-5835	250ml
PluriQ ES-DMEM Medium (MEF Media)	GlobalStem	GSM-2001
DM1 Media Components
D-MEM/F-12 (1X), Glutamax liquid, 1:1	Invitrogen	10565018	385ml
Knockout Serum Replacement	Invitrogen	10828028	20% 100ml
Pen/Strep	Invitrogen	15140122	5ml
Glutamax 200mM	Invitrogen	35050061	5ml
MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid	Invitrogen	11140050	5ml
2-Mercaptoethanol (1,000X), liquid	Invitrogen	21985023	900ul
DM2 Media Components
D-MEM/F-12 (1X), Glutamax liquid, 1:1	Invitrogen	10565018	500ml
Glutamax 200mM	Invitrogen	35050061	5ml
Pen/Strep	Invitrogen	15140122	5ml
N-2 Supplement (100X), liquid	Invitrogen	17502048	10ml
DM3 Media Components
NEUROBASAL Medium (1X), liquid	Invitrogen	21103049	500ml
B-27 Supplement Minus Vitamin A (50X), liquid	Invitrogen	12587010	10ml
Glutamax 200mM	Invitrogen	35050061	5ml
Pen/Strep	Invitrogen	15140122	5ml
Components/Materials
	Millicell	PICM0RG50
Phosphate Buffered Saline (PBS)	ThermoFisher	10010023	500 mL