E173: Optimization of Adrenal Organoid Protocol for Therapeutic Screening

Adrenal and adrenocortical cancer organoids are a preclinical modeling system that can facilitate high throughput experimental therapy screening and enhance our comprehension of the cellular and molecular biology underlying adrenocortical disease. We hypothesized that a robust adrenal organoid culturing platform would develop through 3D culturing techniques and the addition of growth factors known to promote adrenal stem cell diversity.

Methods:

12-week-old male C57BL/6J mice were euthanized and prepped under sterile conditions before performing a midline laparotomy. Adrenal glands were exposed bilaterally by performing a medial visceral rotation and then harvested under a dissecting microscope using ophthalmic scissors and fine forceps. Glands were placed on ice, transferred to a warmed digestion buffer containing DMEM/F12 and 18% collagenase (Sigma, C6885), quenched using DMEM/F12 with 10% FBS and centrifuged. The supernatant was discarded, and the remaining cell pellet was resuspended in 2 ml of  DMEM/F12 and passed through a 70um filter. Cells were counted using a hemocytometer and plated onto 96 well plates coated with 25uL Matrigel or 96 well v-bottom plates with a hydrophobic F108 coating. The following media conditions were tested on a Matrigel-coated plate with Pen-Strep (Gibco, 15140122), FGF (Sigma, F9786) using ~5,000 cells per well: DMEM/F12, DMEM/F12 with 1%, 2%, 5%, 10% embryonic FBS (eFBS), and DMEM/F12 with 10% regular FBS. Six replicates were performed per condition. After 14 days in culture, cells were fixed using 4% PFA and stained using established IHC protocols for Steroidogenic Acute Regulatory Protein (StAR; Invitrogen, PA5-21687) and Nestin (Invitrogen, MA1-110).

Results:

On average, 1x10⁶ cells were derived from five mice. StAR staining revealed that adrenal medullary cells require FBS at high concentrations over serum-free media. Nestin staining showed no statistically significant difference between various media conditions with robust cortical cell growth irrespective of additive growth factors. After 14 days, co-culture of adrenal cells resulted in 3D organoids with evidence of organoid growth over time.

Conclusions:

By optimizing existing techniques for isolating adrenal cells, we increased cell yield 100-fold compared to previous methodologies and optimized a culturing protocol for 3D adrenal organoids. Our results suggest that paracrine signaling between adrenal cortical cells is sufficient to support cell growth in culture. Over time, adrenal organoids reform into a structure resembling a native gland, with cortical and medullary tissues co-localizing.