Application Note

Hypoxic Cell Culture Using Incubator With Precision Low O2 Control

Source: Eppendorf, Inc

By Stacey Willard, Linette Philip and Ma Sha

Oxygen is a critical regulator of cellular homeostasis and as such, oxygen deprivation is lethal to normal cells. In contrast to normal tissues, solid tumors often have regions of significantly reduced oxygenation due to an inconsistent and disorganized blood supply at the center of the tumor. Tumor O2 deprivation, or hypoxia, can result in chemotherapeutic drug resistance and gene expression changes in cancer cells. Decades of cancer research have established that tumor cell growth, survival, motility, the recruitment of blood vessels (angiogenesis), energy metabolism, and cellular differentiation are affected by hypoxia. Patients with tumors displaying high levels of hypoxia often have poor prognoses and negative clinical outcomes. This effect has been documented for many deep tissue tumors such as breast, prostate, ovarian and uterine cancer.

A number of human tumor cell lines have been used to model the effects of hypoxia in an effort to target these drug-resistant cells with new therapeutics. One such cell line, LNCaP, was derived from a bone metastasis from a terminal prostate cancer patient. These cells have been widely used in prostate cancer drug discovery and are uniquely sensitive to androgen (e.g. testosterone and dihydrotestosterone) levels. Danza and colleagues have shown that LNCaP cells previously stimulated with androgens before growth under hypoxic conditions (2 % O2) grow faster than cells maintained in normoxia (20.9 % O2). We chose to use a simplified version of this model to establish a protocol for setting up low-O2 cell culture in a standard tissue culture laboratory. Since standard laboratories do not have access to controlled substances, we did not stimulate the cells with androgen before growth in hypoxic conditions.

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