Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Mar 20;10(3):e0120097.
doi: 10.1371/journal.pone.0120097. eCollection 2015.

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

Teresa M DesRochers  1 , Ivana Y Kuo  2 , Erica P Kimmerling  1 , Barbara E Ehrlich  2 , David L Kaplan  1
Affiliations

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

Teresa M DesRochers et al. PLoS One. .

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Mycoplasma detection and removal.
(A) Top: Representative Western blot of InsP3R3 and PC2 expression after shRNA knockdown. Lanes to the left represent control; lanes to the right represent the knockdown condition. Actin was used as a loading control. Each lane represents a separate sample. Bottom: Quantified analysis of Western Blot. (B) Mycoplasma contamination was detected by PCR before (left lanes), but not after (right lanes) Plasmocin treatment. (C) Mycoplasma was removed from cell lines over 2 weeks by treatment with Plasmocin every other day. Following treatment, the cells lines were grown in antibiotic free media for an additional 2 weeks to promote the growth of any remaining mycoplasma.
Fig 2
Fig 2. Cell proliferation in 2D cell cultures.
Mycoplasma positive and mycoplasma-negative cells were grown for 48 hours and assayed for cell proliferation with an MTT assay. Data represents an average of 8 replicates and the error bars represent the standard error of the mean. The respective p values are: LLC-PK1 = 0.996, InsP3R3 KD = 0.137, and PC2 KD = 0.76.
Fig 3
Fig 3. Cyst formation is improved in 3D tissues without mycoplasma contamination.
Mycoplasma positive and mycoplasma-negative cells were grown in 3D tissues for 2 weeks at which time the tissues were fixed, sectioned, and stained for H&E. Note that the cysts are more diffuse in the mycoplasma-positive tissues. Scale bars = 200 μm.
Fig 4
Fig 4. Cyst number is increased in 3D tissues without mycoplasma contamination.
(A) Mycoplasma positive and mycoplasma-negative cells were grown in 3D hydrogels for 2 weeks at which time the tissues were fixed and stained with Carmine for whole mount imaging. Note that cysts are more numerous in the mycoplasma negative tissues. Scale bars = 100 μm. (B) Quantified number of cysts across 4 different quadrants. (C) The diameter of the cysts is altered upon treatment of mycoplasma contamination. Data represents the average diameter and the error bars represent the standard error of the mean. * represents p>0.05, ** p>0.01, ***p>0.001, ****p>0.0001
Fig 5
Fig 5. Mycoplasma-positive cysts have fewer proliferative cells.
(A). Cysts after 2 weeks in 3D cell culture were stained for an antibody against Ki-67, a marker of cell proliferation (green). Sections were counterstained with actin (using phalloidin red) and DAPI (to denote nuclei, blue). (B) Analysis of the number of cells (as assess by total number of nuclei) per cyst in a cross-sectional area. Analysis is the average of at least 2 different images from three different cyst preparations for each condition, and represents between 9–12 cysts. (C) Absolute number of Ki-67 cells per cyst. Analyses in B and C are the average of at least 2 images from three different cyst preparations for each condition, and represents between 9–12 cysts.
Fig 6
Fig 6. Mycoplasma positive cysts lack organized tight junctions.
Cysts were stained for the tight junctional protein, ZO-1 (green), actin (using phalloidin, red), and nuclei (blue) after 2 weeks of 3D culture. Note that ZO-1 expression is reduced and actin is unorganized in mycoplasma positive cultures. Images are representative of at least two different at least 2 images from three different cyst preparations for each condition. Scale bars = 10 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology. 2002;39(2):75–90. 10.1023/A:1022913015916 - DOI - PMC - PubMed
    1. Torrente M, Guetg A, Sass JO, Arps L, Ruckstuhl L, Camargo SM, et al. Amino acids regulate transgene expression in MDCK cells. PloS one. 2014;9(5):e96823 10.1371/journal.pone.0096823 - DOI - PMC - PubMed
    1. Wayner EA, Brooks CG. Induction of NKCF-like activity in mixed lymphocyte-tumor cell culture: direct involvement of mycoplasma infection of tumor cells. J Immunol. 1984;132(4):2135–42. . - PubMed
    1. Paddenberg R, Wulf S, Weber A, Heimann P, Beck LA, Mannherz HG. Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases. Eur J Cell Biol. 1996;71(1):105–19. . - PubMed
    1. Zhao H, Dreses-Werringloer U, Davies P, Marambaud P. Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminants. BMC research notes. 2008;1:38 10.1186/1756-0500-1-38 - DOI - PMC - PubMed

Publication types

LinkOut - more resources