Determining the Optimal Culturing Conditions of Patient-Derived Colorectal Cancer Organoids using the RealTime-Glo™ MT Cell Viability Assay

In cancer research, standard cell lines and their culturing conditions are not able to properly mimic the parental tumor architecture or its microenvironment. In this context, tumor organoids are of special relevance. Tumor organoids are three-dimensional multicellular structures cultivated in vitro from the tumor stem cells obtained from patient tumor or biopsy. Tumor organoids keep the unique properties of the primary tumor, including drug responsiveness of the original patient’s tumor. Thus, tumor organoids might become a promising tool for decision-making in personalized cancer therapy and also for drug-screenings applications.

The great heterogeneity of the tumors between patients, but also between different areas of the tumor, needs to be considered and reflected by the generated organoids. This is an ongoing challenge. Often the selection of multiple, optimal culturing conditions, for each individual case is required to recapitulate the primary tumor. Therefore, an assay allowing fast and robust selection of the favored media strategies is of great importance to save time and money.

Here we describe a quick and reliable procedure, which allows the selection of the optimal culturing conditions out of a five media strategy for patient-derived colorectal cancer organoids using the RealTime-Glo™ MT Cell Viability Assay.

RealTime-Glo™ MT Cell Viability Assay (Cat.# G9711)
Advanced DMEM/F-12 (Gibco Cat.# 12634028)
Glutamax 100X (Gibco Cat.# 35050038)
HEPES (1M) (Gibco Cat.# 15630080)
Penicillin/Streptomycin (Pen/Strep) 100X (Gibco Cat.# 15070063)
DPBS, 1X no calcium, no magnesium (Gibco Cat.# 14190-094)
EDTA (Sigma-Aldrich Cat.# 431788-25G)
Y-27632 (Abcam Cat.# 120129)
WCM, RCM, NCM (homemade media)
Matrigel (Corning Cat.# 356231)
Nicotinamide (Sigma-Aldrich Cat.# N0636-100G)
N-Acetylcysteine (Sigma-Aldrich Cat.# A9165-25G)
B-27 (Thermo Fisher Cat.# 17504044)
A83-01 (R&D Systems Europe Cat.# 2939)
SB 202190 (Sigma-Aldrich Cat.# S7067-5MG)
Primocin – 500mg (InvivoGen Cat.# ant-pm-1)
m-EGF (REPROTECH Cat.# 315-09)
Gastrin (R&D Systems Europe Cat.# 3006/1) 
Prostaglandin E2 (Sigma-Aldrich Cat.# P0409)
Liberase (Sigma-Aldrich Cat.# 05401135001)
Fetal bovine serum (Biowest Cat.# S1810-500)
Red blood cell (RBC) lysis buffer (Roche Cat.# 11814389001)
GF- (Advanced DMEM/F12 + 1X Pen/Strep + 10 mM Hepes + 1X Glutamax)

Assay procedure to determine which media conditions support tumor organoid growth.

1. Prewarm opaque 96-well plates in the incubator and thaw Matrigel on ice.
2. Wash tumor tissue with 5ml of cold 1X PBS + Pen/Strep 3–5 times.
3. Cut the tissue into small pieces (1–2 mm³) and wash again with cold 1X PBS + Pen/Strep.
4. Transfer the tissue pieces into a 50ml conical tube with DMEM + Pen/Strep + Primocin + Liberase + Y-27632 and incubate for 1 hour on a shaker at 37°C at 250rpm.
5. Pass the fraction through a 100µm cell strainer.
6. Collect the cells in a 15ml conical tube containing 5ml 1X PBS + P/S and centrifuge at 300 × g for 5 minutes at 4°C.
7. Resuspend the pellet in 3ml 1X RBC lysis buffer and incubate 10 minutes at RT in the dark.
8. Neutralize the 1X RBC lysis buffer by adding DMEM + P/S + Primocin + 10% FBS.
9. Pellet the cells by centrifugation at 300 × g for 5 minutes at 4°C, and resuspend the pellet in 3ml of GF-.
10. Count cells and resuspend them at a cell density of 1 × 10⁷ cells/ml.
11. Prepare ice-cold solution with 60% Matrigel and place 10µl drops with 33,000 cells into the center of wells of prewarmed opaque 96-well plates. Also include background control wells without cells (60% Matrigel in GF-).
12. Allow Matrigel to solidify for 15 minute in a cell incubator (37°C, 5% CO₂). Subsequently add 100µl of prewarmed media substituted with 1X MT Cell Viability Substrate and 1X NanoLuc® Enzyme. For details, see the Real Time-Glo™ MT Cell Viability Assay Technical Manual #TM431.
13. Measure the luminescence after 1, 4, 24, 48 and 72 hours of incubation.

Tumor derived organoids are shown from two different patients with colorectal cancer. The cultures are grown out in five different media that, to our knowledge, support the growth of most colorectal tumors. The RealTime-Glo™ MT Cell Viability Assay was started right after seeding the tumor derived cells and was analyzed over a time period of 72 hours. The growth curves established by the signal from the RealTime-Glo™ MT Cell Viability Assay recapitulated very quickly (within hours), revealing which media strategies supported the outgrowth of clones from the tumor tissue. Medium M2, M3, M4 and M5 supported tumor organoid growth from the tumor of patient 1 (P1). Medium M1 did not show satisfying growth support of tumor organoids (Fig. 1).

However, the tumor of patient 2 (P2) showed a completely different outgrowth behavior. Tumor organoids from patient P2 were ideally supported by medium M2 and M4. Medium M1 showed also acceptable growth support (Fig. 2). The cultures in medium M3 and M5 showed very weak growth and were terminated after 72 hours of culture.

These results demonstrate that the RealTime-Glo™ MT Cell Viability Assay can be used to evaluate the metabolic activity of organoid cultures. In this particular application, the assay was used to determine the culture medium conditions that support tumor organoid growth. The main advantage of the assay is that the decision on the medium strategy can be made within 72 hours, a procedure that takes 7 to 10 days by macroscopic evaluation. This saves time as well as money to establish tumor organoid cultures.