Stem Cell Reviews and Reports

, Volume 5, Issue 4, pp 410–419 | Cite as

A Three Dimensional Anchorage Independent In Vitro System for the Prolonged Growth of Embryoid Bodies to Study Cancer Cell Behaviour and Anticancer Agents

  • Chui-Yee Fong
  • Li-Ling Chak
  • Arjunan Subramanian
  • Jee-Hian Tan
  • Arijit Biswas
  • Kalamegam Gauthaman
  • Mahesh Choolani
  • Woon-Khiong Chan
  • Ariff Bongso
Protocols

Abstract

We describe a three dimensional (3D) anchorage independent in vitro protocol for the prolonged growth of human embryoid bodies (EBs) up to 90 days. We grew hESCs (46XX) in methylcellulose (MC) in motion culture in the presence of EB medium (EB), EB medium with Matrigel (EB + MAT), bulk culture medium (BCM), and BCM medium with Matrigel (BCM + MAT). All four experimental groups produced embryoid bodies (EBs) which with prolonged growth to 90 days acquired blood vessels and tissues from all three germ layers. Based on histology, microarray gene expression profiles and the definition for experimental teratomas, we could classify the EBs into early EBs, mature EBs and teratomas. The EB + MAT group produced the highest number of teratomas and their microarray data suggested the presence of inductive microenvironment niches and activation of pathways for self-organization, morphogenesis and growth. When we microinjected hepatocarcinoma-Green Fluorescent Protein cells (HepG2-GFP) (46XY) into the teratomas, after 10 days the HepG2-GFP cells had grown inside the teratoma as confirmed by confocal microscopy and SRY gene analysis. This 3D-MC-(EB + MAT) in vitro system requires few cells to produce many teratomas, can be used to test pluripotency of potential human embryonic and induced pluripotent stem cell lines (hESC, hiPSC), and is an experimental humanized platform to study cancer cell behavior.

Keywords

Embryoid bodies Histology Human embryonic stem cells In vitro teratoma assay Microarray Microinjection 

Notes

Acknowledgements

The authors thank Dr Gan Shu Uin (National University of Singapore) for a gift of the HepG2-GFP cells. This project was supported by funds from the National University of Singapore (R-174-000-089-133) and the National Medical Research Council, Singapore (R-174-000-103-213).

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Copyright information

© Springer Science + Business Media 2009

Authors and Affiliations

  • Chui-Yee Fong
    • 1
  • Li-Ling Chak
    • 2
  • Arjunan Subramanian
    • 1
  • Jee-Hian Tan
    • 2
  • Arijit Biswas
    • 1
  • Kalamegam Gauthaman
    • 1
  • Mahesh Choolani
    • 1
  • Woon-Khiong Chan
    • 2
  • Ariff Bongso
    • 1
  1. 1.Department of Obstetrics & Gynecology, Yong Loo Lin School of MedicineNational University of SingaporeKent RidgeSingapore
  2. 2.Department of Biological SciencesNational University of SingaporeKent RidgeSingapore

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