Home › Cell Biology › Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma
Cell Biology JoVE (Open Access) Citable · DOI

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

DOI: 10.3791/53884-v
What you'll learn
  • ✓ Isolate cancer stem cells from osteosarcoma biopsies using nonadherent culture conditions
  • ✓ Establish and maintain sarcosphere cultures from primary tumor cell lines
  • ✓ Characterize osteosarcoma cancer stem cells using in vitro analysis methods
  • ✓ Verify cancer stem cell identity through molecular and phenotypic markers
Protocol

Biopharma Insights The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

Difficulty
advanced
Total time
~2–3 weeks (initial cell culture establishment plus sphere formation and characterization)
Model organism
Human osteosarcoma primary cells
Biosafety
BSL-2

Steps

1
Establish osteosarcoma cell line from biopsy

Obtain primary cell cultures from human conventional osteosarcoma biopsies and establish adherent monolayer cultures. Culture cells in standard growth medium to generate sufficient cell populations for subsequent stem cell isolation.

▶ 01:20
2
Perform sarcosphere formation assay

Transfer adherent osteosarcoma cells to nonadherent culture conditions to select for cancer stem cells capable of forming three-dimensional sphere structures (sarcospheres). Maintain cultures in stem cell-selective medium.

▶ 03:12
3
Isolate and expand sarcospheres

Collect formed sarcospheres from nonadherent cultures and passage them to maintain and expand the cancer stem cell population. Monitor sphere growth and morphology over multiple passages.

▶ 05:48
4
Characterize isolated cancer stem cells in vitro

Perform molecular and phenotypic analyses on sarcosphere-derived cells to confirm cancer stem cell markers and validate stemness properties. Employ flow cytometry, immunofluorescence, or gene expression assays.

▶ 05:48
5
Verify cancer stem cell culture establishment

Confirm successful isolation and maintenance of verified osteosarcoma cancer stem cell cultures through marker expression, proliferation assays, and functional validation of self-renewal capacity.

▶ 06:24
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