Orca behaviors interacting with humans within apparent friendship bonds are reviewed, and some impediments to the human evaluation of delphinid intelligence are discussed. The subsequent involvement of these orcas and their offspring in aggressive incidents with humans is also documented and examined. This is particularly relevant given that the highest recorded rates of aggressive incidents have occurred among orcas who had previously established unstructured human friendship bonds prior to their inclusion within oceanaria performances. It is concluded that the confinement of orcas within aquaria, and their use in entertainment programs, is morally indefensible, given their high intelligence, complex behaviors, and the apparent adverse effects on orcas of such confinement and use.Based on neuroanatomical indices such as brain size and encephalization quotient, orcas are among the most intelligent animals on Earth. They display a range of complex behaviors indicative of social intelligence, but these are difficult to study in the open ocean where protective laws may apply, or in captivity, where access is constrained for commercial and safety reasons. From 1979 to 1980, however, we were able to interact with juvenile orcas in an unstructured way at San Diego’s SeaWorld facility. We observed in the animals what appeared to be pranks, tests of trust, limited use of tactical deception, emotional self-control, and empathetic behaviors. Our observations were consistent with those of a former Seaworld trainer, and provide important insights into orca cognition, communication, and social intelligence. However, after being trained as performers within Seaworld’s commercial entertainment program, a number of orcas began to exhibit aggressive behaviors. The orcas who previously established apparent friendships with humans were most affected, although significant aggression also occurred in some of their descendants, and among the orcas they lived with. Such oceanaria confinement and commercial use can no longer be considered ethically defensible, given the current understanding of orcas’ advanced cognitive, social, and communicative capacities, and of their behavioral needs. Intelligence is the mental quality that consists of the abilities to learn from experience, to reason, to plan and solve problems, to understand and handle abstract concepts, and to broadly comprehend and adapt to one’s surroundings . CognitiImaging studies have shown that there is a neural distinction between the processing of physical aspects and social aspects of the world, i.e., so called physical and social cognition . PhysicaPhysical adaptation to aquatic life leads to streamlined forms that generally lack the ability to manipulate and carry tools. Added buoyancy reduces striking force while currents and the viscosity of water render the control of objects more difficult. Further, the oceanic environment is three-dimensional with potential tool materials confined to the bottom surface. By its nature, the marine environment lends less advantage for tool use as well as less opportunity for tool creation . While sOn the other hand, social cognition is far more invariant regarding these physical regimes. The neural structures governing instinctive sociosexual behavior have remained all but unchanged in vertebrates for 500 million years , renderiOrcinus orca, occupy all the oceans on Earth, from the Arctic to the Antarctic. Orcas belong to the order Cetartiodactyla. The early ancestors of humans and orcas were already evolutionarily separate in the Cretaceous period, some 90 million years ago. Rapid encephalization within the orca line of evolution developed during the Oligocene period; that within the human line began during the Pliocene period. Orca and dolphin lineages differentiated around 8 million years ago; human and chimpanzee lineages differentiated around 6 million years ago [Orcas, Homo sapiens have existed for approximately 200,000 years [Genetic studies of orca ecotypes  indicate that the root divergence dates back some 700,000 years . Homo sa00 years . Quite a00 years ,12.hideous and that their teeth are the largest and most terrifying in the world [Diving Manual gave orcas their highest danger rating, referring to them as extremely ferocious beasts attacking humans at every opportunity [Orcas have only been maintained in captivity starting in the 1960s when the first captives were studied and displayed in oceanaria. Orca ecotypes with oceanic coastal ranges have a long tradition of powerful totemic association to some native peoples. In Western civilization, orcas have long been considered ferocious killers as exemplified by the writings of Pliny the Elder, et al.  The Natural History of Fishes, 6. The Balæna and the Orca, “This fact however is known to the orca, an animal which is particularly hostile to the balæna, and the form of which cannot in any way adequately be described, but as an enormous mass of flesh armed with teeth”. The Natural History of Pliny, Volume II, Bostok, J. and Riley H. T. (1855). Captain Robert F. Scott, RN, in his 5 January 1911 journal entry on Herbert Ponting’s encounter with orcas at the edge of an ice floe, describes the orcas as he world . As receortunity  (p. 11))www.KinToCetaceans.org.During 1979 and 1980, the San Diego SeaWorld facility  possessed a number of juvenile orcas, aged two to five years that were in training to become performers. They were in their first one to three years of captivity. These orcas were rotated one or two at a time into a marine mammal petting pool (subsequently referred to as the pool) where visitors could interact with them. Authors Anderson and Waayers were among some two dozen regular visitors who established bonds with these orcas. Waayers spent more time with the orcas than Anderson, and the authors include the experiences of others who interacted with the orcas , as well, in the following. Narratives and photographs of the interactions can be found on the authors’ website: The orcas were named: Canuck 2 (subsequently referred to as Canuck), Katina, Kasatka, and Kotar. They were of the North Atlantic ecotype, having been captured in Icelandic waters.Beneath the Surface [This circumstance was rather unique. Orcas who had experienced one to three years of socialization in wild pods were able to form friendship bonds with humans of their own choosing and largely free from supervision. Current United States and Canadian laws forbid human interactions with and constrain approaching wild orcas. Increasing concern over aggressive and sometimes deadly human-orca incidents have led to occupational safety regulations limiting human interactions with captive orcas. Captive orcas are legally held to be property, and they are monetarily valuable. Worldwide there are over 50 orcas in captivity, of which SeaWorld Parks and Entertainment owns around 30. There are corporate protocols for their handling, training, and use in performances, described in former SeaWorld trainer John Hargrove’s book,  Surface   by the non-human may implicitly evoke the corresponding emotion in the human (depending on circumstances), without the time delay of reflection. Humans’ non-conscious perception of emotional stimuli such as facial expressions is well established, and its neuroanatomy is of ancient lineage . Non-humHuman visitors at the petting pool were restricted to leaning against an outer pool wall of waist height. The orcas could rest their heads on or completely slide out onto an underwater shelf extending inward from this wall. Human visitors could then contact the orcas’ heads, including the insides of their mouths, and their entire bodies if the orcas slid onto the shelf fully. These untrained orcas repeatedly sought human physical contact including face and head rubs, tongue rubs, and whole-body rubdowns. Typically, there was little supervision of the interactions between human visitors and the occupants of the petting pool. The following subsections describe additional interactions that are suggestive of social intelligence in orcas. The authors have no personal recollection of incidents in which a petting pool orca harmed a human visitor. They also did not observe mistreatment of the orcas by visitors. Once regular interaction began with an orca, the authors’ subjective interpretation was that the orca appeared to exercise caution such that human harm was avoided. It appeared that the orcas learned which of their behaviors resulted in negative human responses and avoided those behaviors in future interactions. If their motive was to experience positive interactions with humans , they indeed achieved this.Examples of possible orca modification of behavior to mitigate human fear include: (1) During the initial meeting between Anderson and Canuck, Anderson was wary of the orca’s massive tooth-filled mouth, and consciously kept his hands well clear of it. Canuck notably did not close his mouth until well clear of Anderson’s hands; (2) Kotar repeated this behavior on Anderson’s next visit. It was noted that after Anderson felt comfortable placing his hand inside the orcas’ mouths that they halted this behavior; (3) Kotar gently mouthed Anderson’s hand only after about 15 min of tongue rubbing and manipulation.Orcas did sometimes close their mouths on human hands or forearms. Anderson noted that an orca only did this if the human had freely placed his or her hand in the orca’s mouth, and there was always ample time for removal of one’s hand if desired. The authors never observed an orca grasp a hand from mid-air. Also, when an orca did pull on a human hand, it always let go before actually pulling the person into the pool . Waayers noted that Kasatka was the most prone to “snappishness”, but her style involved an open-mouthed swipe near a person’s hand, with no actual harm done, interpreted by Waayers as a “leave me alone” signal.The authors noted what appeared to be a correlation between the level of trust a human showed an orca and the subsequent depth of the orca/human rapport. Seeming tests of trust on the part of the orcas included: (1) Canuck, when first encountering Anderson, suddenly and without warning emerged from beneath a bottlenose dolphin with which Anderson had been interacting, with the orca opening his mouth wide very close to Anderson’s face. Anderson interpreted this as a greeting combined with a test of trust; (2) Kotar greeted Anderson (upon his last visit with the orcas) by sliding on top of him as he bent over the pool wall, and holding him pinned until the author gave him a human style hug in return, seemingly mixing a test of trust with prank-like behavior; (3) Hockins reported Kasatka and Kotar simultaneously pulling on each of his arms, apparently mixing a test of trust with prank-like behavior. Anderson’s assessment is that the issues of trust, humor, and affection often seemed intertwined in the orcas’ actions. This could of course relate to the difficulty of a human interpreting orca intentions. Additionally, Hargrove  (p. 99) There could be alternative explanations for the seeming trust/rapport connection. It is possible that human visitors that were less fearful and more inventive in their interactions with the orcas were seen to be more stimulating and thus more desirable for the orcas to interact with.A tactical deception is an act from the normal repertoire of one individual, deployed such that another individual will likely misinterpret what the act signifies, to the advantage of the first individual . MitchelOrcas did not appear to display this level of tactical deception in the authors’ experience, other than its possible application for humor, as described in the next section. It should be noted that Bigg’s or Transient orcas are silent when hunting marine mammals , which MThe orcas seemed inclined to play pranks, or in other words to display behaviors that appeared to have the sole intent of generating a certain desired reaction in a human or humans. Pranks and tactical deception both contain an element of instilling false beliefs; however, pranks have a humorous intent as opposed to deception’s intent to take advantage.Tactical deception has been noted as a potential indicator of Theory of Mind (ToM) in apes . ToM is Prank-like behavior observed among the orcas included: (1) Kotar sliding on top of the waist high outer pool wall and then squirting the crowd with a mouthful of water only after curious pool visitors approached him closely, as observed by Waayers when a tour group was present; (2) Waayers observed Kotar and Kasatka alternately beaching themselves on the lower pool ledge, as if to request a rubdown. As the closest orca was approached, it slid back into the water, and the other would beach itself further away, with the orcas never allowing an approach close enough for touching; (3) Katina beaching herself on top of Hockins’ forearms, holding him pinned. He reports that she made eye contact with him and subjectively assessed her message to be “I got you”. Whether this was a true “prank” or a demonstration of power is not clear. Hockin’s personal assessment was that it related to trust.The greeting ritual is a form of focused attention following an absence. Most people have likely experienced the greeting rituals of a family dog or cat. Such greetings appear to be a mutual validation facilitating social cohesion. Incidents apparently related to orca greetings of humans include: (1) During the first visit in which Anderson directly interacted with him, Kotar was initially on the far side of the pool, scanning the surroundings in a spy-hop position, and his gaze had briefly fallen upon Anderson, moved on, and then snapped back on Anderson in a sort of “double-take” style. He then swam directly to Anderson, bypassing several other visitors around the pool’s edge. Presumably, Kotar had been the other orca in the pool on Anderson’s previous visit and had observed his interactions with Canuck. Not long after this initial greeting, Kotar presented Anderson with a fish, as described in ○Cognitive ToM which relates to taking the perspective of another.○Affective ToM which relates to comprehending feelings in another.Cognitive empathy has components of:Affective empathy involves internalizing and mirroring the feelings of another.Some orca behaviors seemed suggestive of empathy. There are various types of empathy based in separate neuroanatomies and which are separately activated :CognitiThree examples below may provide evidence of empathy on the part of the orcas:(1) Hockins describes an incident in which Kotar seemed to perform a test of human frailty on him. At first, Kotar bit down hard enough on Hockins to induce pain, and Hockins signaled Kotar to stop. Kotar eased the bite pressure but did not release Hockins. Then Kotar slowly reapplied the bite pressure. When it reached the limit of comfort, Hockins repeated the stop signal. Kotar let Hockins go, but (as interpreted by Hockins) registered surprise at the difference between the two bite pressures. This could be interpreted as reflecting cognitive ToM, affective ToM, and possibly affective empathy. Hockins notes that thereafter, none of the other orcas in the pool ever bit down harder than this level that Kotar established, although none of the other orcas performed this sort of test on Hockins.(2) The authors were first drawn to the orcas in part by a desire to bond with a “fellow being”, and quickly learned that feeding them fish from the trays sold at the pool was not an effective way to achieve this. Many visitors did feed the orcas fish, and it was the authors’ impression that the orcas were in a moderately hungry state, at least while the pool was open to the public, as they readily accepted and ate fish from most visitors. Typically, the orcas would stay with most visitors just long enough to accept fish, and then move along to the next visitor. However, the orcas seemed to spend longer periods of time interacting with people who did not feed them. The authors interpret this as an effort on the orcas’ behalf to interact with certain humans at a level of friendship (as defined in (3) Hargrove   As observed by Anderson and Hockins, sometimes Kotar would interact with them for several minutes, then swim around the pool perimeter in search of offered fish, and then return for more non-food related interaction. He would repeat this cycle many times. On his first meeting with Anderson, Kotar saved the last fish from a circuit of the pool and presented it to Anderson. He repeated this behavior until Anderson understood the fish was a gift and accepted it; (2) When Hockins was being tugged on simultaneously by Kotar and Kasatka, this demonstrated coordinated actions between themselves while simultaneously interacting with a human; (3) Similarly, these two orcas alternately beaching themselves to request a rubdown but then not allowing Waayers to touch them suggests similar mental coordination.Coordinated teamwork has been well documented and might be expected from a social predatory species such as the orca , and it The petting pool orcas were all captured in near coastal waters off southern Iceland in two successive years. It appears that Katina, Kasatka, and Kotar may have been captured on the same day by the same boat and crew and possibly were from the same pod. Some of the records on this are unclear. When the petting pool orcas became performers, they shared a tank with an orca named Kenau, who was captured one year prior, also off Iceland. Winston, another performing whale, was a Resident ecotype captured off the U.S. state of Washington, and so was the only non-Icelandic orca in contact with the petting pool orcas when they became performers.The Icelandic orcas could have reasonably had prior cultural commonality as well as a not too distant familial relationship, particularly Katina, Kasatka, and Kotar. To whatever extent orcas are able to communicate, there is a good chance they had at least related dialects.There was a degree of coherence in the behavior of these orcas that would seem to indicate they were sharing information, some of which was fairly complex. Some instances of directly coordinated behavior have already been noted as in Kasatka and Kotar simultaneously pulling Hockins toward the pool and alternately enticing Waayers for a rubdown.Hockins noted that after Kotar performed a bite pressure test on him, no orca ever bit down harder than the limit Kotar had established as acceptable. Anderson can confirm that the three orcas who mouthed him all applied a uniformly firm but not painful pressure. As best as he can reconstruct, Anderson’s first such experience happened in the same month as Kotar’s test on Hockins but he cannot definitively say which experience occurred first.Hargrove reported Kasatka, who was clearly the dominant orca in the “pod” when he worked with her, according to his interpretation of the events: (1) communicating with another orca that she was not visually able to see, causing that orca to reject a fish reward; and (2) inducing another orca that had been given a salmon to pass the salmon to Kasatka through a gate  (p. 95).Pushing trainers about in the waterMouthing, sometimes to the extent of leaving permanent bite marks, and even breaking bonesLunging at staff who approached the waterPulling trainers into the water and holding them underA substantial minority of SeaWorld orcas have responded aggressively towards their trainers and other staff members. Recorded incidents include such behaviors as:The petting pool orcas and some of their offspring exhibited such aggressive incidents at a noticeably higher rate than any other grouping of incident prone orcas. The next highest rate of such incidents occurs in the group of orcas who were tank mates of the petting pool orcas during the time of visitor friendships. These tank mates were full time performers who had previously completed initial training. There are no indications that they had spent time in the petting pool. The petting pool orcas trained alongside them and did some initial public performances with them. An examination of this history potentially provides further insight into orca social cognition.When these petting pool orcas later completed performer training, the unstructured interactions with human friends stopped. The orcas were shipped among the various SeaWorld facilities and lived the lives of full time performers. Within a few years, these orcas started to become aggressive. Hargrove  (p. 58) In early 2013, a documentary titled “Blackfish” premiered at the Sundance Film Festival in Park City, Utah, USA. It was subsequently purchased by Cable Network News and after some limited audience showings, it was publicly aired in late 2013 and has been shown many times since. Blackfish primarily focuses on the story of an orca named Tilikum who killed SeaWorld trainer Dawn Brancheau in 2010. Tilikum had previously been involved in the deaths of two other humans. Blackfish reviews some of the history of orca captivity and Tilikum’s earlier life at a marine park named Sealand of the Pacific in British Columbia, Canada. All this is presented against the backdrop of other aggressive human-orca and orca-on-orca incidents at SeaWorld. SeaWorld contends that Blackfish conveys falsehoods and employs emotional manipulation techniques. These differing perspectives have polarized public opinion regarding orca captivity.Death at SeaWorld [Similar to an actual historical investigation, one is forced to utilize the surviving documentation and testimonies of the events to attempt to construct a rational account. As the authors do not have original source documentation, they have had to depend in some cases on the work of others. The orca incident data used in the following is based on the work of Stephan Jacobs . He has SeaWorld  (p. 356)As of this writing in May 2016 and since their founding in 1964, SeaWorld has owned 65 orcas of whom 29 (45%) have been involved in one or more published aggressive incidents. A small number of orcas account for the greatest number of incidents. The orcas with greater numbers of incidents appear to fall into five recognizable groups by background and/or ancestry. Having divided a small number of orcas into these groups, they are so few in number that no statistical significance can be claimed, only interesting tendencies. Summing the SeaWorld orca incident data by year reveals another interesting aspect: there are no published incidents during the petting pool years (1979 and 1980) or the following two years. Of the six incidents in 1983 and 1984, four are attributed to the petting pool orca Kandu 5 and one to tank mate Kenau. See About 55% of all of SeaWorld’s orcas have no published aggressive incidents. Of those with no incidents, nine lived beyond puberty and into at least early adulthood. An additional 15 were within the age bounds of puberty. By comparison, almost all of SeaWorld’s orcas with aggression lived to be sexually mature adults, i.e., 26 mature, 2 in puberty, and 1 pre-puberty. It should be noted that some orcas have recorded aggressive incidents starting in pre-puberty: Kayla, Keto, Orkid, Tilikum, and Taku. Kandu 5 had two recorded incidents at the very nominal beginning of puberty. Other than Tilikum, these orcas are all petting pool orcas or their offspring, or tank mate offspring, or joint Tilikum-petting pool offspring. Thus 26 (28) orcas with incidents lived to maturity (mature + in-puberty), while 9 (24) orcas without incidents lived to maturity (mature + in-puberty). This supports a possible correlation of orca aggression to adult versus juvenile behavior and to captive living environment. However, these correlations do not appear as strong as the correlation to having experienced unstructured interactions with visitors. The resultant aggression could still result from the combination of factors.Of additional note, Kona 2 was captured off Iceland along with Kandu 5 and Canuck 2. She was processed with them prior to transfer to SeaWorld; however, she was instead sent to the Orlando SeaWorld facility for training. The same was true of Kahana. She was captured with Katina, Kasatka, and Kotar. She too was sent to Orlando for training.While it is possible to discover a number of independent references to the San Diego orcas interacting with visitors, the authors have not been able to discover any reference to such a practice at Orlando. It is fairly certain that Kona 2 and Kahana did not have the opportunity to develop unstructured relationships with visiting humans. Kona 2 and Kahana both died post-puberty with no published aggressive incidents in spite of having shared the same capture and initial handling. Their cases demonstrate the contrapositive.Hargrove  notes thLatham and Mason  examinedThe question then arises in the case of the petting pool orcas as to what the particular enrichment might have been. The presence of similar behavior in the offspring and tank mates of the former group of orcas potentially indicate cultural transmission of enrichments or the knowledge that they once existed.Alternatively, increased exposure to humans at a young age may have reduced wariness of orcas around humans at later times in their lives. If this were the case, it would apply to first generation orcas exposed to enrichment, and possibly second generation orcas if this reduction in wariness was culturally transmitted.Much of the public and academic discussion about animal welfare has centered on the ethical concerns that arise when humans cause suffering to sentient beings. In the case of delphinids, additional questions arise regarding the level of ethical considerations humans owe to those animals closest to humans in cognitive complexity and capacity .A significant impediment resides in the fact that humans and delphinids have distantly separated evolutionary histories. While the adaptive evolutionary logic of social life was common to both, the evolutionary solutions were often unique, especially in that the solutions were subject to the peculiarities of terrestrial versus marine environments.Marino  discusseThe Encephalization Quotient (EQ) provides one measure of intelligence. The Consistent with Marino’s note on shared mammalian subcortical neuroanatomy, the authors of this paper found orcas quite emotionally familiar, as orcas seem to have done with humans. Additionally, the authors have begun addressing Marino’s question on those more abstract levels as described above with orcas. The quest to objectively compare delphinid cognitive complexity and capacity (or “intelligence”) to that in humans is however made difficult by the non-overlap of capabilities that humans consider to be benchmarks of intelligence.Often, comparisons of human and non-human ToM abilities involve false beliefs; e.g., deceptive behaviors in chimpanzees regarding the location of some food item and the potential knowledge of a competitor regarding that food . AnotherOrcas appear to practice only low-level deception, as in silent hunting by Bigg’s orcas. The authors’ only observations of deception in orcas related to their tendency towards humorous pranks.Orcas practice food sharing among themselves. They do not cache food or other objects. They do not accumulate or carry about objects, except very temporarily in their mouths. As apex predators, they have little necessary concern about other species stealing their food.Orcas have excellent eyesight, but much of their lives are spent in murky waters and sometimes at depths where lighting is greatly reduced. They are highly dependent on echolocation, a sensory mode that humans scarcely possess . The phyOf note, the acoustic pulses of echolocation pass through solid objects that would be opaque to light. The echoes occur at material boundaries where the properties of acoustic transport change. Some acoustic energy is reflected while other energy continues to propagate through the new material.Unlike humans, orcas have two modes of perception with quite different characteristics regarding whether an object would be hidden behind another. Any comparison of ToM and other complex cognitive abilities in humans and orcas needs to consider that behaviors or environmental factors critical to one species might be moot to the other.Dolphins have been studied since the 1960s but certainly less extensively than chimpanzees. Their physically impressive cousins, orcas, have been even less studied. As explored earlier in this paper, orcas and humans have significant overlaps in emotion and social cognition. Humans and orcas can develop friendships. While the friendships developed in 1979–1980 were unstructured, they still enabled significant insights into orca social cognition. If human-orca friendships were approached in a more integrated fashion, for instance, a cultural immersion such as Jane Goodall experienced with chimpanzees, it is possible that far more could be learned.SeaWorld announced in March 2016 that it would cease breeding its orcas and no longer have orcas performing in theatrical shows. SeaWorld stated in a press release, “As society’s understanding of orcas continues to change, SeaWorld is changing with it”. Since the release of Blackfish and facing a continuing campaign by People for the Ethical Treatment of Animals (PETA), SeaWorld’s attendance and stock prices have been suffering.As two of the original petting pool orcas still survive, potential friendships could be revived, and those two orcas could form the basis for extending the friendships to other orcas. The authors suggest that a resumption of treating the orcas with basic respect and rebuilding trust are necessary first steps.Establishing communications with delphinids in some form of common language would be a fundamental breakthrough. Direct human-delphinid communications could lead to more popular acceptance of delphinids as deserving moral consideration, more than any other revelation. Researchers have been seeking this goal since the 1960s. While advances have been made, incontrovertible results have been elusive. The authors suggest that achieving communication breakthroughs could well depend on thinking about how information might be encoded utilizing the physically possible means available to delphinids. Humans encode speech on a single channel that is amplitude and frequency modulated. Information is presented sequentially such that a syntax is required to determine the meaning of word sequences, e.g., for English: subject, verb, and object.Delphinids have at least three channels. They have two phonic lips that can generate different sound types either completely independently, e.g., whistles and clicks, or in phase conjunction with each other  , but also that the limits of their intelligence are not yet clearly defined. We would be similarly wise to appreciate that orcas’ rise to intelligence preceded that of modern humans by more than twice the time that our own species has existed. In fact, orcas were very likely among the most dominant predators in the world’s oceans, before


Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of  Chiroptera which is divided into two suborders Megachiroptera and Microchiroptera, a total of 1,240 species have been yet described Bats belong to one of the most abundant, diverse and widely distributed mammalian groups. In the order of in vitro studies derived from European bat species is desirable. So far, several bat cell lines were reported in previous studies, but most of them were established from non-European bats, like Tb1-Lu from Tadarida brasiliensis, Mvi/It from Myotis velifer incautus, and several primary immortalized cell lines from Pteropus alecto, Carollia perspicillata, Eidolon helvum and Rousettus aegyptiacusEptesicus serotinus employed to investigate the type I interferon (IFN) response after lyssavirus infection Since all of 52 identified European bats species are endangered and strictly protected, the use of animal trials for the investigation of immune mechanisms in bats is not possible. Thus, development of stable cell lines for M. myotis, evaluated their susceptibility to EBLV-1, EBLV-2 and RABV infection and investigated innate immune gene responses after the polyinosinic:polycytidylic acid (poly I:C) stimulation. The established M. myotis cell lines present a valuable in vitro model to study the interactions between lyssaviruses and their natural host, and to shed light on the mechanisms of resistance in bat's central nervous system (CNS).In this study, we established different cell lines from the European bat M. myotis specimen in the Moravian Karst in November 2012 was in compliance with Law No. 114/1992 on Nature and Landscape Protection, and was based on permit 01662/MK/2012S/00775/MK/2012 issued by the Nature Conservation Agency of the Czech Republic. Established M. myotis cell lines from the single sacrificed specimen have been used to examine bat responses to the infection by Pseudogymnoascus destructans (un-published data) as well as for the present study of rabies. Three co-authors of the present manuscript concerning establishment of M. myotis cell lines to investigate lyssavirus infection, i.e. Hana Bandouchova, Jiri Pikula, and Jan Zukal, examine white-nose syndrome in the Czech Republic and hold the necessary permits. A paper based on these permits and excemption from Law No. 114/1992 on Nature and Landscape Protection of the Czech Republic allowing euthanasia of up to 10 M. myotis bats has already been published Ethical approval for all of the capturing and sampling were confirmed by the competent authorities in the respective Federal Republic of Germany and Czech Republic. The Czech Academy of Sciences Ethics Committee reviewed and approved the animal use protocol No. 169/2011 in compliance with Law No. 312/2008 on Protection of Animals against Cruelty adopted by the Parliament of the Czech Republic. The capture and sampling of a M. myotis male was captured in Sloupsko-Sosuvske caves of the Moravian Karst . The bat was kept to minimize stress and handling between capture and euthanasia in a clean plastic box with soft mesh to enable roosting under temperature of hibernation torpor of 6°C and transferred to our laboratory at Veterinary and Pharmaceutical Sciences Brno (Czech Republic) within a day. It was anesthetized to insensitiveness using isofluranum , and then euthanized by decapitation and subjected to necropsy in order to collect organs and tissues. Tissues were freshly isolated from the euthanized bat, and then minced and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin 100 units/mL and streptomycin 100 mg/mL (Sigma). Primary cells were cultured in 6-well plates till the confluence reaches 50–70%. Immortalization was done by transfection of pRSVAg1 plasmid expressing Simian Vacuolating Virus 40 large T antigen (SV40T) with lipofectamine 2000 according to the protocol (Invitrogen). Immortalized cells were expanded and stock frozen. After several passages, the mRNA expression of SV40T (in the established lines) was tested by reverse transcription PCR (RT-PCR) using SV40T specific primers A single M. myotis cell lines derived from brain (cerebrum) (designated MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol), a M. myotis-specific PCR was developed. An NADH dehydrogenase subunit 1 (ND1) gene (Genbank accession number: DQ915043) from M. myotis was used as a species specific molecular marker. The genomic DNA from different cell lines was isolated by DNeasy Blood & Tissue Kit (Qiagen). The concentration and purity of genomic DNA were determined by Nanodrop (Thermo). PCR was performed using a specific primer pair ND1-F and ND1-R  at the Friedrich-Loeffler-Institute, Germany.To confirm the identity of the established nd ND1-R  and genoM. myotis cell lines and the induction of IFN mediated signaling, poly I:C was used to stimulate the cells. Different cell lines were seeded in 24-well plates at a density ranging from 1.2 to 2×105 cells/well, and cultured as described above. Around 20 hours after seeding, cells were transfected with poly I:C (Sigma) at a concentration of 10 µg/mL by lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Twenty four hours post stimulation, cells were harvested into RLT buffer (Qiagen) for RNA extraction by an RNeasy mini kit (Qiagen).To evaluate the IFN response of MmBr and MmTo cells that were infected with a serial MOI of 0.01, 0.1, and 1.0 were harvested at 24 hpi and used for RNA extraction. To confirm the infectivity in later passaged cells, different immortalized cell lines of more than 15 passages were infected with lyssaviruses RABV, EBLV-1 (E. serotinus isolate) and EBLV-2 (M. daubentonii isolate) at a MOI of 0.1. The infected cells were cultured as described above. Cells were collected for RNA extraction at 24 hpi and quantitative real-time PCR (qRT-PCR) was performed on the CFX96 TouchDetection System (Bio-Rad) using SensiFAST SYBR one-step kit (Bioline) according to the protocol. Immunofluorescence analysis was performed on fixed cells using FITC conjugated anti-rabies monoclonal antibody (SIFIN) at 72 hpi as described before MmBr and MmNol cell lines were infected with EBLV-1 at a MOI of 0.01 to set the sensitivity at a Ct value of 22 for the inoculation dose. The viral supernatant was either changed or not changed with fresh medium at 1 hpi, and viral replication levels were measured by qRT-PCR over 72 hpi.Early after immortalization, the third passage immortalized cell lines were used to check the infectivity of RABV. Cells were infected with RABV  at a MOI of 10. Twenty four hours post infection (hpi), infected cells were fixed and permeabilized as described above and visualized by fluorescence microscope. http://bioinfo.ut.ee/primer3-0.4.0/ and http://www.ncbi.nlm.nih.gov/tools/primer-blast/. Primers of target genes and internal control β-actin were listed in −ΔΔCt method qRT-PCR was introduced to measure the mRNA expression levels of immune related molecules in response to poly I:C stimulation and virus infection. The selected molecules include IFN induced genes: IFN stimulated gene 56 (ISG56), ISG43, myxovirus resistance 1 (Mx1) and IFN induced protein with tetratricopeptide repeats 3 (IFIT3), and pattern recognition receptors (PRRs): toll-like receptor 3 (TLR3), retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA+5). All of these primers were designed based the sequence resources from our own un-published sequence database and public databases of bat species. The softwares for primers design include primer premier 5, online tools: MmBrBecause the MmBr is derived from the CNS, the target of fatal infections by lyssaviruses, a further characterization of cell type of MmBr is desired to improve the understanding of the antiviral defense in the CNS. The expressions of cluster of differentiation (CD) 68, a marker for cells of macrophage lineage Molecular characterization of the All data were presented as means ± S.D. Statistical significant differences were analysed by one-way ANOVA using the SPSS software package.M. myotis cell lines brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) were successfully established by transformation with SV40T gene integrating into the chromosomal DNA. Varying cell morphologies were observed in the cell lines, with MmBr, MmTo, MmNep and MmNol being fibroblastic-like, and MmPca being epithelial-like  were observed in MmTo, MmPca, MmNep and MmNol compared to MmBr, respectively, while for MDA5 about 30-fold  or about 6-fold  (p<0.05) higher expression levels were measured (MmBr (p<0.05)  .MmBr, MmNep and MmNol and 8-fold in MmPca (p<0.05) increase in the TLR3 expression, whilst no change in MmTo (MmBr to as high as more than 9000-fold in MmNol (p<0.05)   . The expectively . IFIT3 w(p<0.05) .M. myotis cell lines is their susceptibility to lyssavirus infection. At an early stage of immortalization, cell lines displayed a significant susceptibility to RABV (MOI of 10 at 24 hpi) as demonstrated by the infection with GFP fused RABV. Notably, MmBr exhibited considerably lower viral load compared to the other cell lines  than the other four cell lines measured by qRT-PCR at 24 hpi . The expression of all three PRRs remained mostly unchanged in MmTo, while it was significantly regulated in MmBr with 2-fold increased expression of TLR3, about 25-fold increased expression of RIG-1 and MDA5 at MOI of 1.0 (p<0.05) .To further evaluate the cell line models for study of the different susceptibility between (p<0.05) . A compand IFIT3 . ISG56 mMmBr but not in the other four cell lines  can also be investigated by using these models in the future studies. Preliminary results indicate a correlation between the observed varying susceptibility and the ability to up-regulate the PRRs and the IFN induced genes. Emerging evidences have shown that PRRs play pivotal roles in antiviral immunity in the CNS MmBr, the high up-regulations of RIG-1 and MDA5 revealed activation of RIG-I-like receptor pathway during RABV infection. As previously reported, RIG-1 is a major PRR to induce IFN in the RABV infected cells, and MDA5 may function to sustain the IFN induction MmBr indicate that the production of IFN was induced by activated RIG-1 and MDA5. In contrast, the low expression level of TLR3 implies a vague involvement of TLR3 in anti-RABV infection immunity or resistance. It was shown that TLR3 participated in and benefited the RABV pathogenesis in human neuron cells in vitro. To reach a successful infection, the viruses must overcome the barriers of innate immune system. It was reported that IFN production and signaling pathways were antagonized in P. alecto cell lines under henipavirus infection E. serotinus brain cell line MmBr contrasts to the high viral load and a silent expression pattern of antiviral effectors in MmTo, providing an evidence of a countermeasure to IFN system by lyssavirus in the peripheral tissue versus a protective mechanism to infection in the brain tissue of bats. Microglial cells are one of the major cell populations in the brain tissue. Additionally, comparing to neurons, they can be infected by different RABV strains to a lesser extent MmBr support a microglia-like feature of MmBr in the CNS. It was reported that a mouse microglia cell line can activate strong innate immunity during RABV infection MmBr demonstrated a critical role of microglia in the anti-rabies defense in bat's CNS. In addition to the function of microglia, the clearance of infected viruses in the CNS requires systematical responses through the complex interactions of different brain resident cells. Herein, the establishment and identification of a microglia-like cell model is a first step towards understanding of the complex reactions of CNS in response to lyssavirus infection in the reservoir species. Overall, this preliminary study using established cell lines implies that immune mechanisms that control the virus replication are present in the CNS of bats. It seems that the ability to control the pathogenic RABV replication via IFN system in the CNS contributes to the asymptomatic outcome in bats.First, the existence of the viral sensors TLR3, RIG-1 and MDA5 in these permanent cell lines suggests a capacity of these cell lines to sense a broad range of RNA viruses. The increased expression of dsRNA receptor TLR3 and IFN induced genes ISG56, ISG43, Mx1 and IFIT3 after stimulation with poly I:C mimicking a viral infection indicates that these cell lines can be used as effective M. myotis displaying a variable susceptibility to different lyssaviruses will serve as a useful model to study virus-host interactions and antiviral resistance mechanisms in the ‘natural’ Lyssavirus host. This study provides a preliminary insight into the antiviral innate immunity correlated to CNS against neurotropic viruses infection in bats.In conclusion, the established immortalized cell lines from the European bat


Cell lines are the foundation for much of the fundamental research into the mechanisms underlying normal biologic processes and disease mechanisms. It is estimated that 15%–35% of human cell lines are misidentified or contaminated, resulting in a huge waste of resources and publication of false or misleading data. Here we evaluate a panel of 96 single-nucleotide polymorphism (SNP) assays utilizing Fluidigm microfluidics technology for authentication and sex determination of human cell lines. The SNPtrace Panel was tested on 907 human cell lines. Pairwise comparison of these data show the SNPtrace Panel discriminated among identical, related and unrelated pairs of samples with a high degree of confidence, equivalent to short tandem repeat (STR) profiling. We also compared annotated sex calls with those determined by the SNPtrace Panel, STR and Illumina SNP arrays, revealing a high number of male samples are identified as female due to loss of the Y chromosome. Finally we assessed the sensitivity of the SNPtrace Panel to detect intra-human cross-contamination, resulting in detection of as little as 2% contaminating cell population. In conclusion, this study has generated a database of SNP fingerprints for 907 cell lines used in biomedical research and provides a reliable, fast, and economic alternative to STR profiling which can be applied to any human cell line or tissue sample. Human cell lines and patient-derived tissue are an essential resource for biomedical research. As the need for these resources grows in industry and academia, so have examples of biosample mix-ups –5. The sS1 Table). These consisted of 820 genetically distinct lines. 170 samples consisted of cell lines with one or more related cell lines within the panel derived from the same patient, termed synonymous  or misidentified  or derivative . STR and the SNPtrace Panel profiles for all lines can be found in S1 Table.907 cell lines were used in this study , with redundancy to ensure that a sample can be sex-typed if one assay were to fail. The 90 autosomal loci include 46 loci that are polymorphic across all populations  and 44 aDNA was extracted from frozen cell pellets ranging from 3e6 to 5e6 cells using either DNeasy Blood & Tissue  or 96 Blood & Tissue Kit  following manufacturer’s protocol. DNA concentration was determined by NanoDrop 8000 and normalized to 50ng/ μL.S5 Table). After thermal cycling, the specific target amplification reaction was diluted 100 times and was used for the SNPtrace Panel genotyping and identification as described below. Sample preparation: 10 min. Machine time: 1 h 15 min.All samples were pre-amplified in 96-well plates as described in the Fluidigm SNP Genotyping User Guide. Briefly, all samples were pre-amplified in 96-well plates with the 10X SNPtrace Panel specific target amplification (STA) primer pool, prepared by pooling 2 μl of all 96 pairs of 100 μM STA and 100 μM locus specific primer (LSP) primers diluted with DNA suspension buffer to a final concentration of 500 nM for each primer. In a DNA free hood, 60 μL of 10X SNPtype STA Primer Pool was combined with 300 μL of Qiagen 2X Multiplex PCR Master Mix  and 90 μL of PCR-certified water to create the STA pre-mix. Next, 3.75 μL of the STA pre-mix was combined with 1.25 μL of genomic DNA and thermal cycled with a 15 minute 95°C hot start and 14 cycles alternating between 95°C for 15 seconds and 60°C for 4 minutes  pool and 8 μL of each locus-specific primer (LSP) for each of the 96 SNPtrace Panel assays into a 96 well plate with 29 μL of DNA suspension buffer. Next, 1 μL of each assay was combined with 2.5 μL of 2X Assay Loading Reagent  and 1.5 μL of PCR-certified water creating 5 μL of the assay mix and 4 μL of each 96 assay mixes was transferred to the assay inlets. Following this, 2.5 μL of the 1:100 diluted pre-amplification reaction (see above) was combined with 3 μL of Biotium 2X Fast Probe Master Mix , 0.3 μL of 20X SNPtype sample loading reagent , 0.1 μL of 60X SNPtype Reagent , 0.036 μL of ROX  and 0.064 μL of PCR-certified water. Finally, 5 μL of each sample was transferred to the sample inlets of the Fluidigm High Precision 96.96 Genotyping integrated fluidic circuit (IFC) for genotyping.S5 Table. The data from the IFC were initially analyzed on the Fluidigm SNP Genotyping Analysis Software. Sample preparation (96-samples): 30 min mixing, 10 min transferring samples to IFC. Machine time (96-samples): 20 min priming, 1 h 30 min loading, 1 h 30 min thermal cycling/reading. Data analysis ~30 sec/sample (clean run) or 10 min/sample (more complex samples).Once the samples and assays were transferred, the IFC was inserted into the Fluidigm IFC Controller, HX, which created 9,216 reaction chambers. The IFC was thermal cycled and end point imaged on the BioMark HD System under conditions outlined in All human cell lines used in this publication were processed by Genetica DNA Laboratories  for authentication testing using analytical procedures for DNA extraction (see above), polymerase chain reaction (PCR) and capillary electrophoresis on a 3130xl genetic analyzer (Applied Biosystems). The thirteen core CODIS short tandem repeat (STR) loci plus PENTA E and PENTA D, and the sex-specific locus, amelogenin, were analyzed using the commercially available PowerPlex 16HS amplification kit (Promega Corporation) and GeneMapper ID v3.2.1 software (Applied Biosystems). Appropriate positive and negative controls were used concurrently throughout the analysis. Sample preparation (16 samples): 30 min, 1 min (transfer). Machine run time (16 samples): DNA amplification, 2 hr; genetic analyser, 1h; Data analysis ~30 sec/sample (clean run) or 10 min/sample (more complex samples).2 x shared / . The identity score is 0 if and only if no common alleles are seen at any locus; it is 1 if and only if the exact same alleles are seen in both samples at all loci. Note that this approach does not assume diploid genomes or biallelic markers, nor does it require that the same set of markers be available for every pair of samples.For either SNP or STR data, we followed Tanabe et al.  and compFig. 1B). The distribution of these scores was seen to be approximately Gaussian, so non-synonymous pairs were scored based on the upper tail probability of a normal distribution with the computed mean and standard deviation.Mean and standard deviation for the pairwise identity scores were computed for all synonymous pairs  application (version 2011.1) with the default settings.In brief, we applied a modified version of the PICNIC  algorithSample sex determination was made using both genotype and copy number data. The AA/AB/BB genotype calls were analyzed to determine the fraction of heterozygous SNPs (AB genotype) on chromosome X. The raw, pre-segmentation copy number ratios  were analyzed to determine the average value on chromosome Y. Visual inspection of histograms of these two values each indicated bimodal densities. Cutoffs of < = 0.05 for the heterozygous fraction and > 0.5 for chromosome Y copy number ratio were selected to identify male samples .The genomic regions (hg19) covering the genes PCDH11Y (chrY:2988462-5587151) and PCDH11X (chrX:88455396-92368901) were excluded from the copy number analysis as they generally show locally increased copy number in putatively male samples. These regions makes up the “PAR3” pseudo-autosomal region  of chromCell line SNP data has been deposited in the European Genome-phenome Archive (EGA). Accession number EGAS00001000610.To test the sensitivity of detecting cross-contamination, we created cell mixtures, THP-1:SK-MEL-1, AU565:SK-MES-1 and MCF-7: LOX-IMVI at fixed ratios of . Cell counts were determined by ViCELL (Beckman). DNA was extracted from the cell mixtures using the DNeasy Blood & Tissue Kit  following manufacturer’s protocol. DNA concentration was determined by NanoDrop 8000 and normalized to 50ng/ μL for all DNA samples.Contamination experiments for diploid samples were performed by mixing a human male and female sample. Three pairs were analyzed using DNA supplied by RUCDR Infinite Biologics . Both male and female samples were diluted to 60ng/μL. Contaminated samples were created by mixing each 60ng/μL sample into the other to create 20%, 15%, 10%, 5% and 1% contamination by volume.Metaphase slides were prepared from cell lines that were cultured, harvested, and fixed with methanol:acetic acid (3:1), according to standard cytogenetic procedures. Seven microliters of denatured SkyPaint probe , Vista, CA) was added to each denatured metaphase slide, which then was covered by a glass coverslip and incubated overnight in a 37°C humidified chamber. Slide pretreatment and post-hybridization washes were performed according to the standard supplied protocol (ASI) with slight modifications. Image acquisition was performed with a COOL-1300 SpectraCube camera (ASI) mounted on an Olympus BX43 microscope using a SKY optical filter (ASI). For each cell line, a minimum of 10–15 metaphases were analyzed using the HiSKY v6.0 software (ASI).FISH probes were prepared from purified BAC clones RP11-91A13 and RP11-71M14 (Yq11.221) and RP11-452D1 (17p13.1/Yq12)  and labeled with Orange-dUTP and Green-dUTP , respectively, by nick translation. The Yq11.221 FISH probe is specific for chromosomal locus Yq11.221, while the 17p13.1/Yq12 FISH probe hybridizes to chromosomal locus 17p13.1 and also the entire band Yq12, which generates a huge FISH signal on Yq12. Metaphase slides were prepared from 7 of the cell lines previously harvested for SKY analysis . The metReverse DAPI images were acquired through the FISHView analysis software by inverting the DAPI to a black and white image, which allows for visualization of chromosome centromeres and bands similar to G-banding.S1 Table). Of the samples run, there were 907 individual cell lines of which 820 represent unique individuals. The remaining samples consist of cell lines known to be either derived from the same patient (termed “synonymous”), misidentified or derivatives of parental lines. To evaluate the accuracy with which the SNPtrace Panel can discriminate between related and unrelated cell lines, pairwise comparisons were performed for all samples , synonymous/derivative pairs of cell lines  were also highly similar  and thus could be identified by the SNPtrace Panel . For the samples under consideration, a cutoff of ≥ 90% identity was sufficient to discriminate between the majority of related and unrelated samples . To assess the discriminatory power of 96 SNPs compared to smaller numbers of SNPs, we randomly selected 24 and 48 autosomal SNPs from the SNPtrace Panel, performed pairwise comparisons for each group and compared the distributions of these identity scores with the full panel. As expected, there was a decrease in the variability of identity scores for unrelated pairs as more SNPs were used, indicating an increased discriminatory power [Fig. 1A). Specifically, although the mean identity score for unrelated pairs changed negligibly when SNP counts were reduced to 48 or 24, standard deviation (SD) increased significantly .The SNPtrace Panel simultaneously assesses 96 SNPs, consisting of 90 autosomal and 6 allosomal SNP assays, and interrogates 96 samples in a single run. In total, including biological and technical replicates, we analyzed 968 samples using the SNPtrace Panel obtaining an autosomal call rate of 99.34%, with just 574 out of 87,120 autosomal SNP “No Calls” . This analysis identified some unexpected findings outlined in Table 1, including identification of two previously unknown pairs of identical cell lines (NCI-H2691/NCI-H2803 and KM-12/PMFko14), and identification of unique profiles for a pair of lines previously thought to be synonymous . We also found high concordance, as measured by the SNPtrace Panel, between four lines reported as synonymous  [STRs are the ANSI standard for cell line authentication ,13,32. WFig. 1B) ,34, whicFig. 1B) –37. We aKlijn et al. In press, Nat Biotech) and (iii) the SNPtrace Panel with annotated sex calls. All three assays performed similarly, identifying 40–45% of males as females but only 2–7% of annotated female cell lines as male with a high degree of concordance between assays . No evidence of Chr Yq was found in 4 of the 7 male lines tested which typed as female . In NCI-H522, we detected 1 copy of Chr Yq12 (der(15)t) by SKY and FISH, suggesting that Chr Y was present but what remains of this portion of Chr Y apparently is not detectable by the other methods. Although SKY indicated no presence of Chr Y in NCI-H441, FISH and reverse DAPI banding detected a normal Chr Y in 18% of cells, explaining why the PCR-based STR and the SNPtrace Panel assays detect Chr Y. On closer inspection, the Illumina SNP arrays did, in fact, detect a low level of Chr Y, but this was missed by the cut-offs we used to identify males . The positive control, NCI-H460, showed 1–2 copies of Chr Y per cell by SKY, FISH and reverse DAPI banding.Unlike other reported SNP profiling panels –31, the n assays . The higThese data suggest that many of the cancer cell lines derived from male patients which profiled as female may have lost the entire Y chromosome, not just the amelogenin locus, or may have dramatically rearranged genomes with fragments of Chr Y translocated to other chromosomes.Fig. 2A). We performed the same test on three pairs of tumor cell lines and found that sensitivity ranged from 2%-10% depending on the ratio of the samples, and equivalent reciprocal mixes were often not detected with the same degree of sensitivity. For example, MCF-7 was only detected at 50% contamination (a 50:50 mix ratio in the MCF-7:LOX-IMVI mixture), whereas just 2% contamination of LOX-IMVI was detected (a 98:2 mix ratio in the MCF-7:LOX-IMVI mixture) . Similar results were observed using STR analysis of these samples (data not shown). Our best interpretation of this data is that the degree of aneuploidy and copy number at any given locus may influence the sensitivity of the assay and thus make contamination by a high copy number cell line easier to detect than contamination by a line with fewer copies.Cell line cross-contamination is an acknowledged and major problem in biomedical sciences , and STRDNA-based authentication is required to validate the results of any research using human biosamples. However, there is a remarkable lack of attention to the problem of cell line misidentification by researchers . Some joSTR analysis is the ANSI standard for profiling cell lines. A wealth of cell line STR profiles are available for comparison , and a growing number of organizations offer fee-for-service STR profiling . As an alternative, SNP-based profiling has been suggested as a cost-effective, simple alternative . Here weSimilar to others, we found that SNP-based profiling can detect intra-species cross-contamination as low as 2% with a range of 2–10% , which cS1 Fig.). Such discrepancies required cytogenetic analysis which clearly showed that loss of most or all of Chr Y was occurring. A more detailed cytogenetic analysis of cell lines and the tissue of origin (if available) is required to formally prove if this is a common genomic feature for cell lines in culture, or represents a worryingly high level of poorly annotated cell lines. Our data suggest that Chr Y loss is due to genomic instability as residual Chr Y fragments were found in some samples. While it is clear that it is challenging to accurately determine the sex of a genomically unstable cell line, the autosomal calls are definitive for identifying the origin of a cell line.There is a high rate of calling male samples female by STR ,39. WhenIn summary, we demonstrated that the SNPtrace Panel is a DNA profiling technology which could be used alone or in conjunction with established cell line authentication methods for labs to continually monitor cell line identity. We have provided a resource of 907 SNP profiles for future comparisons and have demonstrated that SNPs provide a fast, reliable, accurate and cost-effective method for assessing cell line or human biosample identity and intra-human cross-contamination.S1 FigA) A549 has normal sex chromosomes for a male. The theta values indicate the absence of heterozygous alleles on chromosome X, with the exception of the PAR1 and PAR3 regions (PAR2 is covered by only one SNP on the array). On chromosome Y, theta indicates the presence of one copy of chromosome Y. (B) NCI-H1045 has normal female sex chromosomes. Theta indicates clear evidence for heterozygous alleles on chromosome X and the signal on chromosome Y is characteristic of homozygous deletion or the lack of chromosome Y. (C) NCI-H441 shows no evidence of heterozygous alleles on chromosome X. The signal on chromosome Y is intermediate between that of (A) and (B), suggesting a mix of cells with one chromosome X and one chromosome Y and cells with just one chromosome X.Theta, a measure much like the B-allele frequency, gives a measure of the relative abundance of the so-called "A" and "B" alleles. A value of ~0.5 indicates heterozygosity. ((TIF)Click here for additional data file.S1 TableIndex: simple numerical identifier; Sample Count: Number of times the sample was run; Replicate type: TR = Technical Replicate, BR = Biological Replicate,— = unique sample; Cell line name: name of cell line; CLID: unique identifier used within Genentech (this identifier can be used to compare with other published data from Genentech); Species: species from which the cell line was derived; Sex: annotated sex of cell line; Primary Tissue: the tissue from which the cell line originated; Tissue Diagnosis: Type of disease the cell line was derived from; Synonymous/derivatives: known synonymous or derivative cell lines; Number of synonyms in dataset; the count of any synonymous/derivative cell lines present in the data set related to this sample; # No call; the number of autosomal no calls for the SNPtrace Panel results. All headers for the SNPtrace Panel and STR data indicate the SNP or STR identifiers.(XLSX)Click here for additional data file.S2 TableTable describing the detailed karyotype of 19 cell lines and the inferred sex type.(XLSX)Click here for additional data file.S3 TableTable describing the detailed results of Chr Y FISH for 7 cell lines.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Characterization of these cell lines showed luminal origin with expression of epithelial specific antigen and cytokeratin 18 and presence of microfilaments and secretary vesicles, microvilli, tight junctions and desmosomes on ultra-structural analysis. Both the cell lines showed anchorage independent growth and invasion of matrigel coated membranes. Karyotype analysis showed aneuploidy, deletions and multiple rearrangements in chromosomes 7, 9, X and 11 and isochromosomes 17q in both the cell lines. P53 mutational analysis revealed no mutation in the coding region in both the cell lines; however NIPBC-2 cell line showed presence of heterozygous C/G polymorphism, g.417 C > G (NM_000546.5) resulting in Arg/Pro allele at codon 72 of exon 4. Screening for mutations in BRCA1&2 genes revealed presence of three heterozygous polymorphisms in exon 11 of BRCA1 and 2 polymorphisms in exons 11, and14 of BRCA2 gene in both the cell lines. Both the cell lines showed presence of CD 44+/24-breast cancer stem cells and capability of producing mammosphere on culture. The two triple negative breast cancer cell lines established from early onset breast tumors can serve as novel  Breast cancer is the leading cause of cancer deaths among women, accounting for 23% of the total cancer incidence and 14% cancer deaths globally . YoungerCell lines provide an important experimental tool in cancer research with major benefit of infinite supply of a relatively homogeneous cell population that is capable of self-replication which can be widely distributed to facilitate comparative studies. Cell cultures established directly from human tumors serve as unique models for studying and manipulating the potentially relevant molecular and cellular processes underlying malignant disease and identification of novel biological therapeutic targets. Majority of breast cancer derived cell lines are from secondary tumours and pleural effusions of patients with advanced stage breast cancers -18. Few 2 and 95% air. Growth factors were gradually withdrawn from the primary cultures after their purification and were presently being maintained in DMEM medium supplemented with 10% FBS and 2 mM Glutamine. Once the cultures were 80- 90% confluent, the cells were trypsinised with 0.05% trypsin and split in a ratio of 1:3 in fresh DMEM medium. Also, the cells were sampled and frozen periodically at various passages. Both NIPBC-1 and NIPBC-2 were completely purified and passaged for 85 and 66 times respectively and characterised thoroughly.Primary cultures were established from tumour tissue obtained from breast cancer patients, who underwent modified radical mastectomy (MRM) or tru-cut biopsies at Safdarjung hospital, New Delhi, India. A part of biopsy was used for frozen section/paraffin embedding to confirm the diagnosis and presence of tumor cells in it. The tumour tissue was collected in DMEM supplemented with antibiotics . The tissue was then washed with PBS supplemented with antibiotics, minced into 1-2 cu.mm pieces removing blood, fat and fibro connective tissue used for enzymatic disaggregation. Enzymatic disaggregation was carried out by incubating the small tissue pieces with 2.5% crude trypsin for 30 minutes at 37°C and with collagenase (0.15%) overnight. Cells released after enzymatic treatment were tested for cell viability using trypan blue and then seeded on to tissue culture flasks and maintained in DMEM medium supplemented with 10-20% FBS, Glutamine 2 mM, and growth factors such as epidermal growth factor (5-15 ng/ml) insulin 100 U/ml. The cells were then maintained in DMEM medium supplemented with 10% FBS and growth factors at 37°C in a humidified atmosphere of 5% CO(i) ImmunoflourescenceFor immonocytochemical analysis cells were grown on cover slips, fixed with methanol-acetone/ 4% paraformaldehyde and incubated with monoclonal antibodies for Estrogen receptor (ER), Progesterone receptor (PR), HER2/neu, Pan-Cytokeratin, Cytokeratins 5/6 and 18, vimentin, epithelial membrane antigen (EMA), Mucin 1 and P53. These cover slips were further incubated with secondary antibody conjugated with FITC (DAKO) and counterstained with propidium iodide (Sigma). Fluorescence was detected using fluorescence microscope.(ii) Flowcytometry6 cells were resuspended in 500 μl of ice cold staining buffer and 3 μl of FITC conjugated Ki67 or primary antibodies of p53 were added and incubated for 30 minutes at 4°C in dark. The cells were then washed and incubated subsequently with FITC conjugated secondary antibodies (for P53 and P21). After incubation the cells were washed twice by centrifugation at 400 g for 5 minutes and resuspended in 1 ml of ice cold staining buffer and kept on ice until analysis. Appropriate isotype controls have been used.Cells were trypsinised to form a single cell suspension, counted and washed with ice cold staining buffer (1× PBS with 3% FBS and 0.05% sodium azide). Approximately, 1× 10For ultrastructure studies, cultured cells were double fixed with 1.2% glutaraldehyde in 0.01 M phosphate-buffered saline (pH 7.5) and with 2.0% glutaraldehyde for l hr at 4°C. Post-fixation was performed with 1% OsO4. The sample was embedded in epoxy resin. The ultra- thin sections were cut (400-500 μ), stained by lead citrate and uranyl acetate and studied under transmission microscope .5 cells/35-mm petri dish was dispersed in a solution containing 0.3% bacto-agar in epithelial culture medium described above. This was then layered over a 0.6% bacto-agar solution in DMEM/F12 supplemented with 10% FBS and fed biweekly with epithelial cell culture medium. Formation of colonies was determined by inverted microscopy for 2-3 weeks post-seeding [The ability of anchorage-independent growth of the purified cancer epithelial cells was determined by growing the cells on semisolid agar at 25th and at 40th passages. A single-cell suspension containing 10-seeding .6/75 cm2 flask. After 48 hrs cells were exposed to 0.1 μg of colchicine (Sigma) at 37°C, for 3 hrs. The cells were then harvested by trypsinization, incubated for 20 minutes at room temperature with a hypotonic solution (75 mM KCL) and fixed with methanol: acetic acid (3:1). Slides were prepared and stained with GIEMSA. G banding was done to analyse chromosomal aberrations [Karyotyping of both NIPBC-1 and NIPBC-2 cell lines has been done at both early passages (P20 and P15 respectively) and late passages (P65 and P52 respectively) to facilitate cytogenetic examination and comparison of the karyotypes between early and late passages. Briefly, cells were plated at approximately 1-2 × 10rrations .5 viable cells were seeded in triplicate in each of six well plates  in DMEMF12 culture medium. Cells were counted in triplicate with a Neubauer chamber at exactly 24 hr intervals for a series of 10 days after staining with trypan blue dye. The growth curve was plotted and the doubling time was calculated from regression equation of the curve [The doubling time of both NIPBC-1 and NIPBC-2 cell lines at passage 35 and 32 respectively, was determined by counting the cell number at regular intervals. On day 0, 1 × 10he curve -31.9 (while simultaneously providing detection of a 1% fraction of mouse contaminant in a human cell line). The eleven loci are amplified simultaneously in a single tube and analyzed by capillary electrophoresis on 3130xl genetic analyzer .STR profiling of NIPBC-1 and NIPBC-2 cell lines was done using StemElite ID System kit (Promega) as per the manufacturer’s instructions. This method allows identification of unique detection of short tandem repeats, seven human STR loci, Amelogenin (for gender identification) and one mouse locus, which include TPOX, vWA, Amelogenin, CSF1PO, D16S539, D7S820, D13S317, D5S818 and MUS. These loci collectively provide a genetic profile with a random match probability of 1 in 2.92 × 104 cells per well. After 12 hours of incubation, invasion chambers were fixed in 4% paraformaldehyde for 15 minutes, stained with hematoxylin, and washed in PBS. Cancer cells that invaded through the matrigel-coated filter on the lower membrane were manually counted under a microscope. Four randomly chosen fields were counted for each well. The experiment was performed in triplicates.To assess the invasive capacity of NIPBC-1 and NIPBC-2 cells, we utilized Corning invasion chambers and coated them with matrigel; MDA-MB-231 cells were used as positive control. Cells were trypsinised and washed twice with PBS before they are transferred to the invasion chamber. Prior to use, chambers were rehydrated with DMEM for 2 hours at 37°C then plated with 5 × 10The mycoplasma DNA was detected by the PCR kit Venor GeM (Minerva Biolabs) as per the manufacturer’s instructions. This kit is specific for a spectrum of contaminants of cell lines and their biological derivatives belonging to Mycoplasma acholeplasma and ureaplasma species. The primer set is specific to the highly conserved 16S rRNA coding region in the mycoplasma genome. Detection requires as little as 1–5 fg of mycoplasma DNA corresponding to 2–5 mycoplasma per sample volume. The resulting PCR products were separated by electrophoresis in 1.5% agarose gel, stained with ethidium bromide, visualized under UV light and documented by photography.et.al.(2006) [The complete coding regions and exon-intron boundaries for BRCA1 gene were screened for DNA sequence variants by automated sequencing on 3130 × l genetic analyzer . DNA was isolated from both the cell lines. 100 ng of genomic DNA was used for PCR amplification with BRCA1&2 specific primers as mentioned in Saxena l.(2006) .Genomic DNA was isolated from NIPBC-1 and NIPBC-2 cell lines using Gene aid DNA isolation kit as per the manufacturer’s instructions. Polymerase chain reaction (PCR) amplified products encompassing exons 2- 3, 4, 5-6, 7, 8, 9, 10 and 11 of the TP53 gene were analyzed for mutations by automated sequencing using 3130xl genetic analyzer .(i) Aldefluor assay and flow cytometryAldefluor assay was performed as per the manufacturer’s instructions  in NIPBC-1, NIPBC-2 and MCF7 breast cancer cell lines. Briefly, single cells obtained from cell cultures were incubated in Aldefluor assay buffer containing an ALDH substrate, bodipy-aminoacetaldehyde , for 30 min at 37°C. A fraction of cells from each sample, incubated under identical condition in the presence of diethylaminobenzaldehyde (DEAB) was taken as negative control . Flow cy+/CD24- breast cancer stem cells by flow cytometry(ii) Identification of CD44+/CD24−/low in mammary tumors has prognostic significance [7 cells suspended in 1 ml of staining buffer were stained with fluorochrome conjugated monoclonal antibodies against human CD44 (PE) and CD24 (FITC) both individually and in combinations. The isotype controls were also added to the cell suspensions as per the manufacturer’s instructions and were incubated in the dark for 45 min. Unbound antibody was washed off and cells were analyzed for the presence of CD44+/CD24- cell population, no longer than 2 hr post staining on BD FACS ARIA. The sorting gates were established using the isotype matched controls [Al- Hajj and collegues have demonstrated that expression of stem-cell markers CD44ificance . Breast controls .This study had been approved by institutional ethics committee of Safdarjung Hospital, New Delhi and consent has been obtained from each patient for participating in this study.We have initiated 31 primary cultures using 44 biopsies obtained from patients diagnosed with carcinoma of breast. Out of these primary cultures, 2 cell lines could be successfully purified and propagated for more than 60 passages. Among the two cell lines established, NIPBC-1 was derived from a breast cancer patient aged 39 years and NIPBC-2 from a 38 year old patient. The breast tumor in both the cases has been diagnosed as Infiltrating breast cancer NOS type, grade IIb and grade III respectively. The two cell lines were established by enzymatic disaggregation followed by differential trypsinization. NIPBC-1 was initiated after enzymatic disaggregation of tumor tissue with trypsin. Cells were seeded onto the surface of a flask and cells which adhered to the surface by day 1, reached confluency by day 10; the cells were then subjected to differential trypsinization, until a pure epithelial cell population is remained in the flask. NIPBC-1 cells are spindle shaped cells which grow sparse, do not grow in layers and adhere strongly to the surface  after 2 weeks, MCF7 cell lines were used as positive control for this experiment which also formed large colonies on agar Figure .Population doubling time of cell lines NIPBC-1 and NIPBC-2 cell lines were determined as described in materials and methods. The doubling time of NIPBC-1 cell line was found to be 33.25 hrs, while that of NIPBC-2 was 31.56 hrs Figure .Karyotype analysis of NIPBC-1 and NIPBC-2 cells has shown that both the cell lines possess aneuploidy. Chromosomes 7, 9, X and 11 showed deletions in various regions in both the cell lines. Cytogenetic analysis has shown multiple rearrangements. NIPBC-1 was near tetraploid with a modal number of 58 to 62 chromosomes, most of the chromosomes exhibited several translocations and marker chromosomes; rearrangements like t(14:15) (q12:q12) and i(17q) were found commonly in these cells; Isochromosomes 17q was the most common aberration identified in NIPBC-1 Figure  and 11. STR profiling of standard STR markers  was doneTo examine the invasion capacity of the two cell lines established in the present study, we have carried out invasion assay on matrigel coated membrane inserts. We found invaded cells on the other side of the membrane upon overnight incubation with chemo attractant (FBS). Breast cancer cell line MDA-MB231 was taken as positive control. The number of cells invading the matrigel matrix are counted in atleast 4 randomly choosen fields per well. The numbers of MDA-MB231, NIPBC-1 and NIPBC-2 cells that invaded through the basement membrane were 527 ± 45.9020, 409.3 ± 32.3161, 290.6 ± 44.2417 respectively Figure a-c. The Various passages of both NIPBC-1 and NIPBC-2 cell lines were tested for the presence of mycoplasma by a PCR based method using “Venor GeM Mycoplasma detection kit”. Both the cell lines have been found to be free of mycoplasma contamination Figure .BRCA1 gene status in the two established cell lines was checked by automated sequencing, no mutation was detected in the coding regions. Further, we found four heterozygous polymorphisms in exon 11 of BRCA1 and two polymorphisms in exons 11, and 14 of BRCA2 gene in these cell lines , at codon72of exon 4, resulting in p.P72R ; While, NIPBC-1 has homozygous Pro/Pro allele (no change), at codon 72 Figure   both showing nonbasal origin. In India premenopausal patients constitute about 50% of all patients. Early-onset breast cancer may, in part, be biologically different from breast cancer patients in older patients [ yrs old reported . The epireported ,42, furt[et al.,  stained [et al., ,46.et al., [Vimentin which was originally identified as an intermediate filament protein present only as an intracellular component has been detected recently on the surface of cancer cells but not on healthy cells in a punctate distribution pattern. Nicole F. Steinmetz et al., ,48 have et al., [et al., [The ultra structural features confirming epithelial origin of both the cell lines include presence of bundles of microfilaments, secretary vesicles, tight junctions, desmosomes, and granular cytoplasm with numerous ribosomes. Both NIPBC-1 and NIPBC-2 cell lines are negative for estrogen receptor (ER), progesterone receptor (PR) and HER2/neu, and hence represent triple negative breast cancers (TNBC), which are considered to be aggressive tumors having higher histological grade, worse prognosis and occur mostly in younger women -51. Mostet al.,  reported[et al.,  reportedNIPBC-1 and NIPBC-2 both formed large colonies on soft agar confirming the transformed nature of both the two established cell lines since the ability to form anchorage independent colonies is related to transformed nature of the cells . FurtherBoth NIPBC-1 and NIPBC-2 cell lines have shown aneuploidy upon karyotyping, which is a feature of neoplastic cells, supporting their neoplastic origin. Further they exhibited several translocations and rearrangements pointing towards their neoplastic nature. The karyotypes of both NIPBC-1 and NIPBC-2 cell lines exhibit isochromosome 17q. The isochromosome 17, i (17q) is a relatively common karyotype abnormality, that results in loss of the short arm (17p) and duplication of the long arm (17q) leading to a single copy of 17p and three copies of 17q, that has been observed in solid tumors such as medulloblastoma, gastric cancer, bladder and breast cancers, associated with tumor development and progression ,57. Thisp53Arg72 and p53Pro72 proteins do not differ in their ability to bind to DNA, they bind to components of different transcriptional machinery, influencing differential susceptibility to cancer [Mutations in tumor suppressor genes BRCA1/2 and P53 genes are significant contributing factors for breast carcinogenesis; hence we have screened these cell lines for mutations/ polymorphisms in these genes. Genetic mutation or allelic polymorphisms in p53 gene are the most common genetic changes associated with much cancer susceptibility in human cancers, such as cervical cancer, breast cancer, lung and colorectal cancer . Althougo cancer ,71. The o cancer ,72. The o cancer ,74.Mutations in BRCA1 and BRCA2 genes are associated with predisposition to breast cancer; hence we have screened both the cell lines for mutations/ polymorphisms in these genes. Several polymorphisms were observed, among polymorphisms in BRCA1 exon 11, g.2201 C > T is a frequent polymorphism reported in US ,76, and et al. using the cell surface marker CD44+/CD24−/low/Lin − and recapitulated the heterogeneity of the original tumor by injecting them into nude mice [+/CD24- breast cancer stem cells respectively which is relatively similar to the commercially available breast cancer cell line. When plated in serum free, non-adherent conditions as described earlier [CSCs have been isolated from many solid tumors, including breast ,79, pancude mice . Both NI earlier , both NIin vitro models to study breast tumorigenesis in young breast cancer patients and identification of unique therapeutic targets.Molecular analysis of the cell lines was done to identify unique properties of the cell lines. Short tandem repeat (STR) profiling was done to ensure the integrity of human cell lines. The STR profile of NIPBC-1 and 2 found to be unique and do not match among themselves and also with other cell lines in the laboratory that are being used, which shows these two cell lines are novel. Although a number of normal and breast cancer cell lines established from western patients are available, there is a paucity of cell lines established from Indian breast cancer patients. So far only two breast cancer cell lines have been established from late onset breast cancer patients (>60 years) using primary tumors , hence tThe authors declare that they have no competing interests.Performed the experiments: SLP. Analyzed the data: SLP and SS. Provided breast tumor tissue for establishment of breast cancer cell lines: NKS, CC, MRS and DB. Helped in purification of cell lines: BSA; Maintainence of cell lines: BSA, SLP; Helped in Karyotype analysis and mycoplasma detection: MK, RD, ML. Helped in P53 and STR analysis: BSA, ML, AS, SM and SSimon. Overall supervision of the project: SS. Contributed reagents/ materials/ analysis tools: SS. Wrote the manuscript: SLP and SS. All authors read and approved the final manuscript.Expression of cell cycle marker Ki67 by FACS in MCF7, NIPBC-1 and NIPBC-2 cell lines along with their isotype controls. (a) MCF7 (b) NIPBC-1 (73.01%) (c) NIPBC-2 (94.11%).Click here for fileSTR profiling of NIPBC-1 and NIPBC-2 cell lines.Click here for fileSequence variations found in BRCA1 and BRCA2 genes in NIPBC-1 and NIPBC-2.Click here for fileList of primers used for p53 sequencing.Click here for file