nanon effects on mammalian cell long-term cultures: a caveat for experimental hematologists dealing...

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LETTER TO THE EDITOR Nanon effects on mammalian cell long-term cultures: A caveat for experimental hematologists dealing with in vitro long-term culture assays This letter aims to make cell culture investigators using human or animal long-term cell cultures aware of potential contamination by nanobacteria-like particles [1–3], recently proposed as nanons [4]. The real nature of these replicating particles has already been demonstrated in numerous studies. Raoult et al. [4], in particular, showed that these particles are self-propagating mineral-protein complexes, the major component of which is fetuin. Moreover, they demonstrated that nanon mono- layer can be suppressed by ultraviolet/gamma irradiation, as well as by contact with either trypsin or ethylenediamine tetra-acetic acid [4]. Although the debate involving both geologists and microbiologists about whether or not nanobacteria can be considered as living particles [5] is ongoing, awareness should be increased among scientists about the potential contamination of cell cultures with nanons. The prevailing presence of nanobacteria in fetal bovine serum (FBS), commonly used in cell biology studies as a fundamental constituent promoting cell activities in standard conditions, at 37 C in a humidified atmosphere of 5% CO 2 in the air [6] was also demonstrated. Use of human and animal hematopoietic cell lines is a central element of our research [7,8]. Acquisition of human and animal cell lines from qualified donors; estab- lishment of new human hematopoietic cell lines; as well as expansion, characterization, and cryopreservation in liquid nitrogen of all the aforementioned cell types are, in fact, integral parts of our scientific activity. During the last month, unexpected cell deterioration during Epstein Barr virus (EBV) immortalization of human mononuclear cells [9] was identified after just a few weeks’ incubation period. A new serum batch, validated by prelim- inary tests according to United Kingdom Coordinating Committee on Cancer Research guidelines for use of cell lines in cancer research, was used throughout the whole period [6]. Manor’s alternative procedure [10] regarding the use of human autologous or nonautologous plasma for EBV-mediated human B-cell immortalization was not taken into account. As suggested by United Kingdom Coordinating Committee on Cancer Research guidelines, we carefully analyzed EBVþ cell culture samples by inverted microscope and, at the same time, applied standard microbiological methods to investigate the possible reasons for cellular dete- rioration [6]. Light microscopy inspection, in particular, showed the presence of multisized particles characterized by Brownian movements, whereas standard microbiological methods were unable to detect any positive result for microorganisms. Bacteria-like aggregate presence in long-term cell cultures has been described in the literature [1–5], which led us to screen a set of serum lots purchased from different companies in order to evidence the possible presence of nanons and test the lot efficacy in eliciting the desired bio- logical response, i.e., cell survival and proliferation by human 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, and AQE23764), Euroclone (Milan, Italy; lot EUS0080692), and Gibco (Carlsbad, CA, USA; lots 4130992 K, 410980 K, 41Q3467- K, 41QQ272 K, 41F8380F, 41G7771 K, and 1185060). Two-milliliter undiluted aliquots from each batch of serum or 20% diluted aliquots in Iscove’s modified Dulbecco’s medium (IMDM) were added to 24 multiwell plates and incubated at 37 C in a humidified atmosphere of 5% CO 2 in air for 2 to 12 weeks [3]. IMDM culture medium alone or Myelocult (lot 05K16939) was incubated under identical conditions and served as control. After 2 weeks of incubation, 11 of 14 (78.57%) undiluted serum batches ap- peared cloudy and exhibited a naked-eyevisible white deposit. A similar cloudy aspect was observed in the same 11 lots of serum diluted in IMDM after 12 weeks of incuba- tion. Interestingly, undiluted and 20% diluted FBS lots EUS0080692, 413092 K, and 410980 K remained negative, even after 12 weeks of observation. IMDM culture medium alone and Myelocult confirmed the nature of negative controls throughout the entire inspection period of 12 weeks. Offprint requests to: Rosa Di Noto, B.Sc., CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Napoli, Italy; E-mail: [email protected] 0301-472X/09 $–see front matter. Copyright Ó 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2009.05.013 Experimental Hematology 2009;37:887–888

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Page 1: Nanon effects on mammalian cell long-term cultures: A caveat for experimental hematologists dealing with in vitro long-term culture assays

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:

[email protected]

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.

Page 2: Nanon effects on mammalian cell long-term cultures: A caveat for experimental hematologists dealing with in vitro long-term culture assays

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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