Nanobacteria-like particles: a threat to cell cultures

Abstract

The main goal of this study is to alert researchers who work with cell cultures for the risk of contamination by structures called nanobacteria (NB). NB are tiny structures with size varying from 80 to 500 nm, commonly occurring in clusters and producing a biofilm which contains carbonate or hydroxyl apatite. The most likely source of cell culture contamination by such organisms is serum used as supplement in culture media. The presence of NB leads to a progressive culture deterioration with accumulation of granules (probably phagocytized NB) in cytoplasmic vacuoles, an increasing number of dead cells in the supernatant and degeneration of cells that remained attached to the bottom of the vessel. NB can also be found in culture supernatants where they are found in clusters with variable size and displaying brownian movement. In this study, 19 cell lineages, 8 batches of sera and 1 batch of growth supplement from different sources were analyzed. Samples from sera were cultured in Eagle's Minimum Essential Medium (E-MEM) or incubated directly at 37ºC. Tests carried out to detect the presence of extracellular bacteria, Mycoplasma sp and viruses were all negative. Analysis by scanning electron microscopy (SEM) revealed tiny oval structures less than 500 nm in size, isolated or in small groups, in all material analyzed except in one fetal bovine serum batch.

MSCs Conditioned Media and Umbilical Cord Blood Plasma Metabolomics and Composition

“…These include chemically defined media, human serum, UCB serum or plasma (hUCBS or hUCBP), human platelet lysate (HPL) and platelet-rich plasma (PRP) [11] . As a matter of fact, the use of FBS is always associated with batch-to-batch variability, unexpected cell growth characteristics, cytotoxicity of uncharacterized factors in the serum, and risk of possible contamination with virus, prions, bacteria, nanobacteria, mycoplasma, yeast, fungi, and endotoxins [13] – [16] . Also, the use of FBS implies a huge number of fetal calves that must be euthanized for blood collection.…”

Humanized Culture of Periosteal Progenitors in Allogeneic Serum Enhances Osteogenic Differentiation and In Vivo Bone Formation

“…Although it allows cell attachment and sustained cell proliferation and differentiation, the use of FBS in culture conditions has been criticized because, in addition to growth factors and nutrients, FBS contains animal proteins and potential infectious agents, thereby eliciting immunological responses [2]. In particular, infectious agents such as prions, bacteria, viruses, mycoplasma, yeast, and fungi can be difficult to remove from the serum, raising concerns about the use of FBS in culture conditions for cells prior to cell therapy [3, 4]. In addition, the presence of xenogeneic proteins in FBS can alter the outcome of cell‐based therapies.…”

Occurrence of Nanobacteria-Like Particles in Renal Stones of a Southern Brazilian Population

“…Stone fragments were deposited on glass coverslides, air dried and fixed with 1.25% glutaraldehyde (Merck) containing 4% sucrose in 0.5 M phosphate buffer solution (PBS) for 16 h at 4˚C. Following extensive washing procedures with sterile PBS and rinsed in deionized water the fragments were dehydrated in a graded series of ethanol and treated with gold layer as described previously [8]. The samples were then examined by SEM (Philips E.M. 301).…”

“…Biomineralization in cell culture medium [8] yielded the formation of biofilms and mineral aggregates very similar to those found in tissue calcifications and renal pathologies, suggesting that NB could be efficient nuclei of mineralization, which start the formation of kidney stones [9]. Increasing number of reports has shown the role of NB in urologic diseases [10] and their occurrence in human renal pathologies such as polycystic kidney disease [5] and stone formations [11].…”