A 3D rotary renal and mesenchymal stem cell culture model unveils cell death mechanisms induced by matrix deficiency and low shear stress.
BACKGROUND
In epithelial and endothelial cells, detachment from the matrix results in anoikis, a form of apoptosis, whereas stromal and cancer cells are often anchorage independent. The classical anoikis model is based on static 3D epithelial cell culture conditions (STCK).


METHODS
We characterized a new model of renal, stromal and mesenchymal stem cell (MSC) matrix deprivation, based on slow rotation cell culture conditions (ROCK). This model induces anoikis using a low shear stress, laminar flow. The mechanism of cell death was determined via FACS (fluorescence-activated cell sorting) analysis for annexin V and propidium iodide uptake and via DNA laddering.


RESULTS
While only renal epithelial cells progressively died in STCK, the ROCK model could induce apoptosis in stromal and transformed cells; cell survival decreased in ROCK versus STCK to 40%, 52%, 62% and 7% in human fibroblast, rat MSC, renal cell carcinoma (RCC) and human melanoma cell lines, respectively. Furthermore, while ROCK induced primarily apoptosis in renal epithelial cells, necrosis was more prevalent in transformed and cancer cells [necrosis/apoptosis ratio of 72.7% in CaKi-1 RCC cells versus 4.3% in MDCK (Madin-Darby canine kidney) cells]. The ROCK-mediated shift to necrosis in RCC cells was further accentuated 3.4-fold by H(2)O(2)-mediated oxidative stress while in adherent HK-2 renal epithelial cells, oxidative stress enhanced apoptosis. ROCK conditions could also unveil a similar pattern in the LZ100 rat MSC line where in ROCK 44% less apoptosis was observed versus STCK and 45% less apoptosis versus monolayer conditions. Apoptosis in response to oxidative stress was also attenuated in the rat MSC line in ROCK, thereby highlighting rat MSC transformation.


CONCLUSIONS
The ROCK matrix-deficiency cell culture model may provide a valuable insight into the mechanism of renal and MSC cell death in response to matrix deprivation.
Shimony
Nilly
N
Cell and Gene Therapy Program, Division of Nephrology, Department of Medicine, Tel Aviv Academic College of Engineering, Tel Aviv, Israel.
Avrahami
Idit
I
Gorodetsky
Raphael
R
Elkin
Gregory
G
Tzukert
Keren
K
Zangi
Lior
L
Levdansky
Lilia
L
Krasny
Lina
L
Haviv
Yosef S
YS
D000818:Animals; D023102:Anoikis / Q000502:physiology; D017209:Apoptosis / Q000502:physiology*; D002292:Carcinoma, Renal Cell / Q000473:pathology; D016923:Cell Death / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival; D002478:Cells, Cultured; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D005109:Extracellular Matrix; D005455:Fluorescent Antibody Technique; D006801:Humans; D008545:Melanoma / Q000473:pathology; D050527:Mesangial Cells / Q000166:cytology* / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology; D051379:Mice; D008962:Models, Theoretical; D018384:Oxidative Stress / Q000502:physiology*; D011336:Probability; D051381:Rats; D017382:Reactive Oxygen Species / Q000032:analysis; D012680:Sensitivity and Specificity; D013314:Stress, Mechanical
D017382:Reactive Oxygen Species