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Cell Biology JoVE (Open Access) Citable · DOI

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

DOI: 10.3791/53685-v
What you'll learn
  • ✓ Set up and operate an enclosed cell culture system for PSC/NSC maintenance
  • ✓ Configure gas concentrations and temperature in a cGMP/cGLP-compliant environment
  • ✓ Execute sterile feeding protocols and culture monitoring procedures
  • ✓ Evaluate pluripotent stem cell derivation outcomes under controlled conditions
Protocol

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Difficulty
advanced
Total time
~3–7 days per culture passage (including differentiation cycles)
Model organism
Human pluripotent stem cells (PSC), human neural stem cells (NSC)
Biosafety
BSL-2

Steps

1
Set gas concentrations and temperature

Configure oxygen, carbon dioxide, and nitrogen levels in the enclosed system. Establish and verify incubation temperature parameters to achieve stable internal conditions.

▶ 00:54
2
Operate buffer chambers

Initiate and manage buffer chamber functions to maintain internal atmosphere equilibration and support consistent culture microenvironment conditions.

▶ 02:34
3
Prepare incubator and facility

Initialize the enclosed incubator system, verify cGMP/cGLP compliance, and prepare the cell production facility work environment for aseptic culture operations.

▶ 03:53
4
Feed and maintain cell cultures

Perform routine medium exchange and feeding procedures for pluripotent and neural stem cell lines within the sterile enclosed system.

▶ 05:36
5
Evaluate pluripotent stem cell outcomes

Assess morphology, viability, and pluripotency markers of PSCs derived under hypoxic conditions using Sendai virus reprogramming.

▶ 06:23
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