The effects of mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts.

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination

Kim-1 measured cytotoxicity in NKi-2 cells and tissues upon treatment with cisplatin, gentamicin, or doxorubicin.

<p>Kim-1 secretion was measured by ELISA in the supernatant of both NKi-2 2D cell cultures and 3D tissues at 0, 3, 7, 10, and 14 days following treatment with (<b>A</b>) 0.01 µM to 1 µM cisplatin, (<b>B</b>) 0.2 mM to 2.2 mM gentamicin, or (<b>C</b>) 0.001 µM to 0.2 µM doxorubicin. n  =  7.</p

NKi-2 cells maintain kidney function when grown in 3D tissues.

<p>(A) Adenylate cyclase activity in NKi-2 cells within the bioengineered 3D tissue upon treatment with ADH or PTH. Treatment with forskolin acted as a control for production of cAMP. n  =  4. (<b>B</b>) Na⁺ dependent glucose uptake in NKi-2 cells within the bioengineered 3D tissues. Tissues were exposed to 2-DG with or without Na⁺ in the form of NaCl. *p<0.05, n  =  5. (C) RT-PCR of drug tranporters found in kidney epithelial cells using RNA from both NKi-2 cells and 4 week 3D tissues. (<b>C</b>) RT-PCR of different transport proteins found in human kidney cells in both NKi-2 cells (2D) and 4 week old 3D tissues.</p

NKi-2 cells formed tubular structures when grown in 3D tissues.

<p>(A) Schematic of 3D tissue formation. An acellular layer of 1:1 Matrigel:rat tail collagen I (1 mg/mL) is layered onto a tran-well membrane. Following polymerization, a layer of the same ECM mixture containing NKi-2 cells is added. The tissues are maintained in growth media and after approximately 2 weeks, the cells organize into branching structures. (B) Morphology of NKi-2 cells after extended growth in 3D tissues. Tissue sections, 10 µm, were stained with H&E and whole tissue sections were stained with carmine for whole mounts. Arrows indicate areas of branching tubular-like structures. Scale bars  =  100 µm. (<b>C</b>) Expression of epithelial cell markers, e-cadherin (red) and cytokeratin 8/18/19 (green), kidney proximal tubule cell marker GGT1 (green) and organic anion transporters OAT1 (green) and OAT4 (red) within the 3D tissues after 4 weeks of growth. Scale bars  =  25 µm.</p

Morphology of 3D tissues after treatment with cisplatin, gentamicin, and doxorubicin.

<p>Tissues were fixed after 2 weeks of drug treatment. (<b>A</b>) 8 µm sections were stained with hematoxylin and eosin to visualize changes in tissue structure. (<b>B</b>) Whole fixed tissues were stained with carmine for whole mount visualization of tissue structure. Low dose refers to the lowest does tested and high dose refers to the highest dose tested. Scale bars  =  50 µm.</p

NGAL measured cytotoxicity in NKi-2 cells and tissues upon treatment with cisplatin, gentamicin, or doxorubicin.

<p>NGAL secretion was measured by ELISA in the supernatant of both NKi-2 2D cell cultures and 3D tissues at 0, 3, 7, 10, and 14 days following treatment with (<b>A</b>) 0.01 µM to 1 µM cisplatin, (<b>B</b>) 0.2 mM to 2.2 mM gentamicin, or (<b>C</b>) 0.001 µM to 0.2 µM doxorubicin. n  =  7.</p

Kim-1 and NGAL secretion in untreated NKi-2 cells and 3D tissues.

<p>Untreated NKi-2 cells were grown for 24 hours followed by media sampling at 0, 3, 7, 10, and 14 days. The media was assayed for the presence of (<b>A</b>) Kim-1 or (<b>B</b>) NGAL by ELISA. NKi-2 3D tissues were grown for 2 weeks followed by media sampling at 0, 3, 7, 10, and 14 days and assayed for the presence of (<b>A</b>) Kim-1 or (<b>B</b>) NGAL by ELISA. **p<0.01, ***p<0.001, n  =  7.</p