Home › Cell Biology › An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates
Cell Biology JoVE (Open Access) Citable · DOI

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

DOI: 10.3791/57922-v
What you'll learn
  • ✓ Culture HEK293 cells in O-shaped vessels using orbital shaking
  • ✓ Generate uniform cellular aggregates with improved homogeneity
  • ✓ Perform medium changes and cell collection from suspension cultures
  • ✓ Assess aggregate morphology and viability by microscopy
Protocol

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Difficulty
intermediate
Total time
~5-7 days (including culture maturation and aggregate formation)
Model organism
HEK293
Biosafety
BSL-1

Steps

1
Seed HEK293 cells into O-shaped bag

Inoculate HEK293 cells into O-shaped vessel at appropriate density for suspension aggregate culture. The specialized bag geometry supports uniform cell aggregate formation during orbital shaking.

▶ 01:18
2
Perform medium change on growing cultures

Replace culture medium while maintaining cell suspension to support continued aggregate growth and metabolic health.

▶ 02:42
3
Harvest and collect cellular aggregates

Retrieve cell aggregates from the culture vessel using appropriate collection techniques for downstream analysis or further culture.

▶ 03:11
4
Assess aggregate morphology and cell viability

Evaluate the size distribution, uniformity, and viability of harvested aggregates using microscopy and viability assays to confirm protocol success.

▶ 04:07
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