nanon effects on mammalian cell long-term cultures: a caveat for experimental hematologists dealing...
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Experimental Hematology 2009;37:887–888
LETTER TO THE EDITOR
Nanon effects on mammalian cell long-termcultures: A caveat for experimentalhematologists dealing with in vitro long-termculture assays
This letter aims to make cell culture investigators usinghuman or animal long-term cell cultures aware of potentialcontamination by nanobacteria-like particles [1–3], recentlyproposed as nanons [4].
The real nature of these replicating particles has alreadybeen demonstrated in numerous studies. Raoult et al. [4], inparticular, showed that these particles are self-propagatingmineral-protein complexes, the major component of whichis fetuin. Moreover, they demonstrated that nanon mono-layer can be suppressed by ultraviolet/gamma irradiation,as well as by contact with either trypsin or ethylenediaminetetra-acetic acid [4].
Although the debate involving both geologists andmicrobiologists about whether or not nanobacteria can beconsidered as living particles [5] is ongoing, awarenessshould be increased among scientists about the potentialcontamination of cell cultures with nanons. The prevailingpresence of nanobacteria in fetal bovine serum (FBS),commonly used in cell biology studies as a fundamentalconstituent promoting cell activities in standard conditions,at 37�C in a humidified atmosphere of 5% CO2 in the air [6]was also demonstrated.
Use of human and animal hematopoietic cell lines isa central element of our research [7,8]. Acquisition ofhuman and animal cell lines from qualified donors; estab-lishment of new human hematopoietic cell lines; as wellas expansion, characterization, and cryopreservation inliquid nitrogen of all the aforementioned cell types are, infact, integral parts of our scientific activity.
During the last month, unexpected cell deteriorationduring Epstein Barr virus (EBV) immortalization of humanmononuclear cells [9] was identified after just a few weeks’incubation period. A new serum batch, validated by prelim-inary tests according to United Kingdom CoordinatingCommittee on Cancer Research guidelines for use of cell
Offprint requests to: Rosa Di Noto, B.Sc., CEINGE, Biotecnologie
Avanzate, Via Comunale Margherita 482, 80145 Napoli, Italy; E-mail:
0301-472X/09 $–see front matter. Copyright � 2009 ISEH - Society for Hemat
doi: 10.1016/j .exphem.2009.05.013
lines in cancer research, was used throughout the wholeperiod [6]. Manor’s alternative procedure [10] regardingthe use of human autologous or nonautologous plasma forEBV-mediated human B-cell immortalization was not takeninto account.
As suggested by United Kingdom CoordinatingCommittee on Cancer Research guidelines, we carefullyanalyzed EBVþ cell culture samples by inverted microscopeand, at the same time, applied standard microbiologicalmethods to investigate the possible reasons for cellular dete-rioration [6]. Light microscopy inspection, in particular,showed the presence of multisized particles characterizedby Brownian movements, whereas standard microbiologicalmethods were unable to detect any positive result formicroorganisms.
Bacteria-like aggregate presence in long-term cellcultures has been described in the literature [1–5], whichled us to screen a set of serum lots purchased from differentcompanies in order to evidence the possible presence ofnanons and test the lot efficacy in eliciting the desired bio-logical response, i.e., cell survival and proliferation byhuman EBV-immortalized B lymphocytes.
We compared 14 lots of serum from five different compa-nies, i.e., Sigma Aldrich (St Louis, MO, USA; lot 067K3395),Lonza (Basel, Switzerland; lot 7SB0006), Hyclone (Logan,UT, USA; lots CSD0413, CMF0158, ALK14879, andAQE23764), Euroclone (Milan, Italy; lot EUS0080692), andGibco (Carlsbad, CA, USA; lots 4130992 K, 410980 K,41Q3467- K, 41QQ272 K, 41F8380F, 41G7771 K, and1185060).
Two-milliliter undiluted aliquots from each batchof serum or 20% diluted aliquots in Iscove’s modifiedDulbecco’s medium (IMDM) were added to 24 multiwellplates and incubated at 37�C in a humidified atmosphere of5% CO2 in air for 2 to 12 weeks [3]. IMDM culture mediumalone or Myelocult (lot 05K16939) was incubated underidentical conditions and served as control. After 2 weeks ofincubation, 11 of 14 (78.57%) undiluted serum batches ap-peared cloudy and exhibited a naked-eye�visible whitedeposit. A similar cloudy aspect was observed in the same11 lots of serum diluted in IMDM after 12 weeks of incuba-tion. Interestingly, undiluted and 20% diluted FBS lotsEUS0080692, 413092 K, and 410980 K remained negative,even after 12 weeks of observation. IMDM culture mediumalone and Myelocult confirmed the nature of negativecontrols throughout the entire inspection period of 12 weeks.
ology and Stem Cells. Published by Elsevier Inc.
888 P. Mirabelli et al./ Experimental Hematology 2009;37:887–888
It is worth noting that all of the aforementioned sampleswere evaluated at 2 and 12 weeks of incubation for micro-biological contamination and no microorganism was de-tected [6].
Finally, other aliquots of the same mononuclear cells, usedpreviously, were thawed and EBV immortalization wassuccessfully carried out using IMDM at 10% v/v of nanon-freetested FBS lots as well as Myelocult complete medium.
We report here that several commercial FBS lots containabundant nanons that can heavily interfere with in vitrostudies using mammalian long-term cell cultures. In thisrespect, human experimental hematology goals, such asmanagement of highly difficult primary cell lines, isolationof desired cells from tissue samples, cloning of cell linesfrom mixed populations, as well as establishment of newimmortalized human cell lines, cannot be achieved in theabsence of alternative, but too expensive, serum-free media.Consequently, in addition to United Kingdom CoordinatingCommittee on Cancer Research criteria suggested for serumbatch testing [6], we propose to apply the described prelim-inary test in order to avoid the consequences of usingnanon-positive sera, which do not adequately support cellgrowth.
The present observation can be relevant not only forexperimental hematologists and basic research investiga-tors, but also for clinical hematologists and oncologists em-ploying long-term assays for evaluating in vitro aspectsrelated to the effectiveness of therapeutic protocols.
AcknowledgmentsWe thank the companies cited in the text, which provided the lotsof sera to be tested for nanon presence.
Conflict of InterestNo financial interest/relationships with financial interest relatingto the topic of this article have been declared.
Peppino Mirabellia,b
Francesca D’Alessioa,b
Elisabetta Mariottia,b
Maria Romanoa,b
Giuliana Fortunatoa,b
Marica Gemeia,c
Rosa Di Notoa,b
Luigi Del Vecchioa,b
aCEINGE, Biotecnologie AvanzatebDipartimento di Biochimica e Biotecnologie Mediche,
Universita Federico IIcEuropean School of Molecular Medicine, Napoli, Italy
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