When to use Primary cells and when to use cell line? Does Passaging change the primary cells' characteristics?

I am working with Bovine Aortic endothelial cells and in those cells we want to check the effect of growth factors on a particular gene at the mRNA levels. There are human primary cells available but I did not find any human endothelial cell line. I wanted to know what are the advantages of using a primary cell over cell line ( I know that cell lines are transformed and are a deviation from normal cells). Also, in terms of the experiment when should we use cell line and when should we check in primary cells?


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  • Neale Foxwell · University College London
    I have worked with BAEC's in the past. It would be interesting to use them again and assess endothelial nitric oxide synthase under the conditions which we grow some of our cells in the COY hypoxic chamber (i.e. at 01-1.0% oxygen).
    As people have previously stated cell culture is a flawed method for looking at what happens to cells in vivo. I am sure that growing cells at 21% oxygen as we all do is not a good starting point.
  • Ewa Gregoraszczuk · Jagiellonian University
    I only partly agree with of my predecessors deductions. From physiological point of view cell line are not useful . For 30 years I worked with coculture of theca and granulosa cells. I insulates them from different sloughterhause animals but always in the same period of the follicular phase. VERIFYING for me is the secretion of estradiol in primary cultures. It showing me that my model is good. However I used one rule. Always are pooled cells from the same size of follicles from 5-6 animals. Then I avoid intra-individual differences. Answering for questions interesting for me cell line of eg granulosa cells is not appropriate.
  • Gaber Ramadan · United Arab Emirates University
    I agree with most of the answers but my recommendations are:
    1. Primary cell culture is better for your work but don't forget the control culture
    2. If you have the ability to do 3D culture, please go ahead
    3. Primary cell culture is very closely related to In vivo testing
    4. if you design to perform your work after passaging the primary cells, try to do that on cells with the same Passage number
  • Nahi Yaseen · Iraqi Centre for Cancer & Medical Genetics Research
    Stability of long term cell ljne depends on the origin of that cell line. This stability usually seen in transformed cell lines (apparantly normal cell line). No cell line can be gown in vitro for long term unless the cells are genetically transformed that escape division control mechanisms. However cancer cell lines usually show different genetic change including chromosomal changes. When we examine colorectal cancer cells for example we found no similarity in chromosome change among cells of the same colorectal cancer type. Milions of changes can be seen in the same cancer type. Primary cell culture is more close to the original cells in vivo, within time the invitro culture conditions will form a pressure on the cells that makes cells change their characteristics to live under those in vitro culture. Any researcher can test this truth by studying the chromosomal situation in any cell line during many serial passages, tgen the results will definitely show changing in the pattern of chromosomal changes indicating characteristic cha nges.
  • Elisabeth Stein · Medical University of Vienna
    Interesting topic and a great conversation that came up!
    I agree on all the points made.
    We work with living organisms and therefore it almost is thier nature to change when exposed to selective conditions as cell culture really is.
    I worked with a great variety of cells so far and sometimes even when you work with the said "well characterized" cell lines that are generally accepted (also by journal reviewers) as model system (in my case bone biology) you can not believe your results until you could also show it in primary cells. AND if you find something that speaks strongly against the use of a certain cell line as model system, don't be too shy to publish it and share this knowledge. We had results that were compromising one of the most used cell lines in our field and it was indeed hard to convince people of our findings.
    I'm aware of the long list of said disadvantages of primary cells, but they are the better model to mimic the in vivo conditions.
  • Wendy Weston · Miami VA Medical Center
    @Shambhavi...I agree with all of the above points, so I will just let you know what we do to address the "cell line vs primary cells" issue. Because the cells we work with are very infrequent, and the assays we use require a significant number of cells to obtain all the results we want....we first do our experiment with a cell line (several batches from different frozen stocks). If possible, we then repeat the experiment in primary isolated cells that we collect ourselves. We only use fresh isolated cells and they are only reliable for ~2 weeks. We try to use them on the same day. I realize that this may not be possible for your studies, but as the great comments made in this post have already pointed out, time is your enemy with cells out of their context (the body). When the cells are removed from the body or even culture conditions that are more biologically appropriate, I have seen changes in their phenotype within hours!!
  • Michael Allman · Aberystwyth University
    Hi All to risk further muddying the waters does anyone have any thoughts on the relationship, pros and cons of explant tissue cultures compared to primary/established cell line lines as ex vivo models?
  • Venil Sumantran · Indian Institute of Technology Madras
    Michael, in my experience, explants of articular hyaline cartilage are preferred to articular chondrocytes for studies on osteoarthritis . We and others published data showing that explant cultures work v. well for evaluating anti-arthritic and anti-inflammatory drugs. This suggests that explants are giving physiologically meaningful results which in turn means that the paracrine signalling in intact. This is not surprising because the ECM of explants is undisturbed. In the case of Articular chondrocytes, there is no authentic ECM and they rapidly de-differentiate in vitro. Hope this helps!
  • Hi there, I agree with Daniel, all cells are part of a system and if their reaction to specific conditions are to be tested surely they have to be tested within a system which is as close to their natural environment as possible. I've been looking at some of the 3D systems for some of the stuff we hope to be doing and it looks like this is the way to go especially when drug testing and clinical trials may be a follow on.
    If you can use explants that may help but as pointed out by most folk you have to get your conditions right very quickly so that you don't lose them, and if the tissue is from human donors they will be rare (make sure that ethics, consent etc is all taken care off.) so practice on the closest animal equivalent is essential to get your technique sorted.. If you can use the 3D systems and get the correct proportions of cells (so cell ID would be important) you could probably grow a nice system which you can use in situ for testing then process for IHC, Immuno or whatever.
  • J W Wang · University Medical Center Utrecht
    Agree with the points above. But one may always keep their minds open: No perfect models exist, regardless of whatever cell lines, primary cells, animal models or even certain groups of patients. Simply because of variation between individuals, species etc combined with environmental factors. Nature is complex. And science is expenditure, we have to start somewhere depends on where you are. And it works, as you see the knowledge gained in the past by our predecessors. Just some common-sense comments.
  • Nahi Yaseen · Iraqi Centre for Cancer & Medical Genetics Research
    In addition to the strong effect of in vitro conditions on cancer cell lines grown in vitro, the freezing down process exert another side effect on those cells. Freezing cancer cell in liquid nitrogen may alter the proportion of cell subclones within the same cancer cell population of the same tumour origin. In spite of all those disadvantages of in vitro cancer cell lines, they remain the best tool for most biological and medical research work.
  • Abraham Alahmad · University of Wisconsin, Madison
    Immortalized endothelial cells may be fine for basic routine stuff (like learning to grow and maintain cell culture) but they are not viable in long term. The advantage of primary are that they are close from in vivo however they differentiate rapidly and loose their original phenotype. There are some described in the litterature but their origin are a bit "exotic" such as the EAhy.926 (hybridoma) or the ECV306 (that some people consider as bladder carcinoma mislabelled as sponatenously immortalized HUVECs).
    I am working with brain endothelial cells and so far the primary rat brain endothelial cells isolated from rat are the best in terms of material access, if you can access to bovine and porcine, it is also fine.
    For primary cell cultures from companies, except HUVECs, I had bad experience overall with them.
  • Nahi Yaseen · Iraqi Centre for Cancer & Medical Genetics Research
    Primary culture can be used to assess the previous in vivo effect of something targeted on the cultured cells while long term culture can be used to assess different in vitro impacts on that cells because primary culture may lives for short term and cannot give the results of in vitro studies like peripheral blood lymphocyte in short term culture. Long term cell lines can be used to assess such as cytotoxicity , cytogenetic changes, physiology and other biological test because these cells stay living for long term and can be followed up easily.
  • Jai Ghosh · Shivaji University, Kolhapur
    Once the primary cells are transformed into normal cells, there will be certain changes from their function point of view. Therefore, checking the effect fo growth factors would be best on the normal cells than on primary cell line. But then it all depends on which is the gene you are trying to study.
  • Venil Sumantran · Indian Institute of Technology Madras
    Cell line data alone is hard to believe, even when many cell lines of the required tissue are used. Primary cultures data is essential in all in vitro studies even though it is difficult to get and maintain. If no primary cells are available, one can use immortalized cells (intermediate between normal and transformed). We and others have reported that immortalized MCF10 cell line gives different responses when compared with normal and transformed human breast cancer cell lines like MCF-7 etc. Unfortunately, it is not easy to find immortalized cell lines for many tissues like ovarian and colon epithelium. Hope this helps!
  • Jai Ghosh · Shivaji University, Kolhapur
    Keeping in view all practicality, i would always say that the data obtained from normal cells is not all that wrong. Of course the inferences from the data should not be an extrapolation to infinity.
  • Kate Lewis · University of California, San Francisco
    I would like to add the additional comment about cell lines, that even if they are obtained from reputable cell banks and are found not to be contaminated, they may behave differently and have a different genetic profile. This is what our group has found using the Walker 256 breast carcinoma cell line from two different cell banks:

    Characterisation of Walker 256 breast carcinoma cells from two tumour cell banks as assessed using two models of secondary brain tumours.

    Kate M Lewis, Elizabeth Harford-Wright, Robert Vink, Mounir N Ghabriel
    Cancer Cell International (impact factor: 1.97). 02/2013; 13(1):5. DOI:10.1186/1475-2867-13-5
    Source: PubMed
  • Adrian Gemiarto · James Cook University
    I would say that choosing one over another depends on what kind of experiments are you planning to do with them. Cell lines obtained from cell repository are either "altered" or cancerous cells, in which they have high number of mutations. This may not go well if you are planning to do some gene expression analysis, due to the eQTL difference caused by some mutations. On the other hand, using cell lines have the upper hand on reproducibility, which is kind of the pitfall of using primary cells (as they behave differently with each passage).
  • Frederic Grosjean · University Hospital of Lausanne
    As mentionned before, whatever you observe in a cell line must be confirmed in a different cell line or ideally in primary cells to ensure that whatever you observed is not linked to a specific mutation that occured in your cell line.

    Most (if not all) cell lines are cancer-derived cells and the more they are passaged, the more mutations they will accumulate. It's always wise to test several "similar" cell lines for whatever you are looking for and then primary cells if it is possible.

    We recently published a paper on dendritic cell lines ( and how usefull they were to screen different treatments, activations,... and then primary cells were used to confirm whatever interesting observation we made in these cell lines. These cell lines showed a significant interest in the fact that we could grown millions without problem, wheras it is difficult to get a lot of primary dendritic cells (linked to the fact that they don't survive very long ex-vivo). It was therefore possible to test a lot of parameters and get a lot of material for large preliminary experiments, before focusing on much lower cell number from primary DC and confirming our results.

    Unless you are working on the specific behavior of a cell line towards a given treatment with increasing passage number (which also would need controls), primary cells are the best tool to confirm your preliminary observations made in the cell lines.
  • Parvaneh Farzaneh · Iranian Biological Resource Center
    Adding all of above answers, it is important to know that in personalaized medicine we should use primary culture for each person and also the most similar results to in vivo studies can be made with primary culteres which are not passaged.
    The origin of cell lines in some cell banks is not from the originators and they prerpared them from the researchers in their countries. unfortunately some of them are missidentified( recently I had received two missidentified cell line from CLS cell bank), so you should order the cell lines from the most reliable cell banks.
  • Ewa Gregoraszczuk · Jagiellonian University
    I agrre with Parvaneh Farzaneh.
    Some years ago I examined the effect of different chemoteraupetyków used in patients with ovarian cancer in primary cultures. I used a test Alama Blue and caspase-3 activity. The answer to the individual used alone or in many cases, two at the same time was different. In some cases it was indeed visible inhibition of proliferation apoptotic effect action, but unfortunately 30% was seen not only the lack of inhibition of proliferation but even stimulation.
    Unfortunately, the number of cases (30) was so small that I could not publish the results, but I have the results in my database
  • Venil Sumantran · Indian Institute of Technology Madras
    Yes, Ewa is correct. We also got respondesr and non-repsonders to Anti-inflammatory drugs in explants of cartilage from arthritis patients. We had small sample numbers and did statistics on these small groupss and showed significant results. When the reviewer rejected the data, we pointed out that there is real heterogeneity in patient response, and how bichemical proof is absent in the literature. Then, our paper got accepted! Ewa, I hope you publish your data...such info is needed for ovarian cancer too ! All the Best!
  • Ewa Gregoraszczuk · Jagiellonian University
    Thank you for this statement. Please send me a pdf of your publication. I will be able cite your research in the discussion.
  • Venil Sumantran · Indian Institute of Technology Madras
    Dr. Ewa, Thanks for your interest in our paper. We worked on herbal drugs and published in a good journal (ECAM). I have attached the pdf. Citing this may not help you for a cancer paper..correct? If you find any on ovarian cancer where they talk of CR, PR (Complete response and Partial response), it may be worth citing them. What about the samples you used for primary cultures? Do you have any patient details on CR and PR for them. That will help a lot. Hope this helps!
  • Ewa Gregoraszczuk · Jagiellonian University
    Dr Venil,

    The aim of our study was to find the both cheap and quick method for selecting the best chemotherapy for individual patient. I had a medical history and histopathological diagnosis.
    Tumor derived tissue were obtained from 11 ovarian cancer patients (not much). Cells were obtained from tumor explants by enzymatic dissociation and incubated with different combination of chemotherapy drugs for 6 days. At the end proliferation and caspases activity was evaluated. 50% had been resistant for both single and multidrug chemotherapy. We received enough cells that can be carried out all the combinations in each female patient
    What's that? used in patients had no effect. But they were also female patient who have had single drug activity. So why give 2-3 simultaneously? I think that 200-300 patients tested so would give chance to take into consideration such examinations prior to treatment.
    I have to once more to look at this set aside publications and try to publish somewhere
  • Venil Sumantran · Indian Institute of Technology Madras
    Yes, it is worth doing more samples and getting reproducible data. Can you use explants without enzyme digestion?? Enzymes may kill some cells-right? As you know, there are companies selling such technology--Oncoprint, Mammaprint, histoculture assay etc...but I do not think they are validated for clinical use -especially in ovarian cancer (OC). If you can choose the most popular drugs for OC, and do expts with 50-100 samples and show response or lack of will be great ! Pl. try for this!
  • Chang Hwang · Asan Medical Center
    My colleagues believe that primary cells are preferred by reviewers and in high impact journals especially with human primary cells. That's why researchers have trouble in choosing cell types for their research and use primary cells as a first choice for higher impact journal publication.
    As I am in tissue engineering and regenerative medicine, a feasibility study of new method need to be produced with cell lines. Reproducibility is main issue at first step and cell lines are better for this purpose. However, cell lines are different from primary cells, it looks the results need to be confirmed with primary cells.
    It looks that clinical translation, primary cell based study should be a choice, but proof of concept or other basic research may start with cell lines.
  • Ewa Gregoraszczuk · Jagiellonian University
    I have I applied trypsin to obtain single cells. The amount of material obtained during surgery is small to perform all variants in several repetitions. In the case of trypsin, cell viability is realy good (90-95%). I trypsinized several times in a very short time. Trypsin activity easily inactivate.
  • Ewa Gregoraszczuk · Jagiellonian University

    I do not agree with you. The cell line is only one cell type. In the organs are important co-operation of all the cells making up him and secreting various factors acting on auto-paracrine manner.
  • Venil Sumantran · Indian Institute of Technology Madras
    Right Dr. Ewa, I hope to see your pdf on this OC work soon. Its very important from personalized medicine point of view. I think Dr. Chang's point re: working out proof of concept in cell lines is understandable because (as you say), we get so little primary tissue. Once we have an interesting result from cell line, we must verify in Primary culture.
  • Vivek Mishra · University of Pittsburgh
    I am totally agree with sakthikumar and stuart. If you want your answer try to read both answer. They have taken care of all the issue. So i dont want to write anymore. Good luck
  • Nahi Yaseen · Iraqi Centre for Cancer & Medical Genetics Research
    Long term established cancer cell line are a good tool for research work in various aspects. However, in vitro studies on cell line showed that cells reveal continuous changes through passages due to the effect of in vitro culture condition. Freezing down of cells for storage reasons affects the cell population which kill some subclone and allows other to overgrow. Therefore you are advised n use primary culture if available which may reflect the properties of cells when they were in vivo.
  • Ping Song · University of Oklahoma Health Sciences Center
    Passaging does change primary cell's characteristics, expecially in response to some gene deletion.
  • Mohammad Yazdi · Tehran University of Medical Sciences
    I think primary cell culture is being used into simulating the in-vivo condition. As a matter of fact ex-vivo culture (isolation of primary cells from the body and cultivate them in culture flasks) is more closer to body real condition than working on line cells. But line cells mostly used in order to check the potency of drug specially in cytotoxicity assays. As far as I know due to the limited time in primary culture (24 -72 h) the cells in this type have no time for change in their characteristics but in line cells, We usually do our assessment between passage number 4 to 8 because of possible change in cells behavior or traits.
    Hope it would be of help.
  • Shaima Salman · McMaster University
    In general, cell lines are mostly homogenous and primary cells are heterogeneous. You really want to use cell lines when you're studying for example molecular signalling pathways since immortalized cell lines are homogenous, easily manipulated and propagated. Once you make predictions about a signaling pathway, you can then move ahead and use primary cell cultures which are more physiologically relevant to test the predictions made using immortalized cell lines. Of course provided that you have access to both...
    Hope this helps,
  • Mayada Hasoon · University of Dohuk
    In my opinion, dealing with growth factors and their effect on particular gene is quite difficult job with primary finite cells, there is no enough time and no enough passages you can do to have pertinent results.
    From my experience, I prefer using continuous cell lines for such kind of work.
  • Ken Tawara · Boise State University
    Like others have said, for a strict study of gene expression in response to growth factors, I would use cell lines over primary cells. This is mainly because gene expression in cell lines are easily reproducible not by just your lab but at other institutions as well.
    Primary cells are useful for other studies from cell differentiation, co-cultures, ECM studies, etc etc. But the problem is that the results are not easily reproducible, and the experiment must be repeated at least 3-6 times more than with cell lines to get statistical confidence in the data. Handling of primary cells can significantly alter their behavior and gene expression, and use of different plastics, serum, brands of media, and even different technicians can all cause the cells to behave differently. If you need to collect primary cells from various individuals, you add another layer of variability and the number of replicates that are needed increases even more.
  • Daniel Medina · Rutgers, The State University of New Jersey

    Your reasoning for use of cell lines for gene expression studies because the data is easily reproducible is a poor reason to use cell lines. Yes, cell lines have their place and have provided a great deal of information on pathways involved is diseases such as cancer. This said, I consider cell lines are a poor model of disease and the data is not a reproducible as you state.

    Cell line behavior is also affected by different culture vessels (type of plastic, coatings etc.), serum, media, technicians, confluency, passage number. If we compare gene profiles from Hela cells grown in my lab with cells grown in your lab we would identify differences. In my experience most labs don’t seem to have SOPs for cells culture and is often conducted in a willy-nilly manner with minimal is any quality controls.

    There is a growing body of data that should motivate us to direct more energy toward the development of in vitro models that would better predict the success or failure of chemotherapeutic agents.
    a. Handling of primary cells can significantly alter their behavior and gene expression, and use of different plastics, serum, brands of media, and even different technicians can all cause the cells to behave differently. If you need to collect primary cells from various individuals, you add another layer of variability and the number of replicates that are needed increases even more.

    If one look at the literature, there are hundreds of papers identifying “predictive” markers using cell lines, yet very little progress in predicting and improving cancer treatment has been made. This makes me wonder how clinically relevant or cell lines model cancer. Just the manner in which cell lines are derived from patient samples raise questions of how well they represent the original tumor. One in culture, the tumor cells go through crisis resulting in significant cell death (>90%). Thus, the cells that survive are only a subpopulation of the original tumor and have been selected for their ability to growth in the hostile environment of the culture flask. In addition, most cancer cell lines have been maintained for decades in monolayer as opposed to three-dimensional culture, and in high oxygen (21%, whereas physiological oxygen tension ranges from ~2% to 5%).

    Recent studies have shown at the genomic level that “driver mutations” are retained in cell lines but drift at the transcriptome level occurs. This data leads to the conclusion that cancer cell lines are more similar to each other, independent of the tissue of origin, than to the clinical samples that they are supposed to model.

    So yes, working with primary cells is more problematic than cell lines, but primary cells better represent the tumor biology and heterogeneity encountered daily in the clinic. This should encourage all of us to step up to the challenge and work of developing better in vitro models.
  • Monica Mattei · Universita degli studi di Ferrara
    In general it might be better to use primary cells. However oftenly it is difficult to have samples, and the number of cells may be limiting. Therefore some studies are difficult to perform with primary cells. In my opinion, cell lines may represent a good in vitro model, although results might be then proved and verified also in primary cells.
  • Hardik Patel · The Feinstein Institute for Medical Research
    Hi Shambhavi,
    I have worked with both primary human endothelial cells isolated from umbilical vein (HUVEC), a cell line (HPMEC-ST1.6R). as per our requirement, we needed a large number of cells for various different experiments. We started with the primary cells, but we did not find consistent results, thus we found this newly developed cell line HPMEC-ST1.6R, that has been working for us. if you choose to use primary cells, make sure that you plan well and try to do all your experiments and replicates around the same time so you will have consistency with passage number and culture conditions.
    here is the article for this cell line:
  • Paul Jones · University of Saskatchewan
    Also do not forget that passaging can also affect 'stable' transformed cell lines. These changes can be a gradual drift over numerous passages or can be abrupt changes in just a few passages late in cell life. We try to standardize as much as possible the number of passages our cells go thru before we discard and start a fresh culture from frozen cells.
  • Anil Tiwari · Postgraduate Institute of Medical Education and Research
    It is a question that depends upon the fashion and publication demand. You can use cell lines for standardization and confirming all the experiments, as all these needs large number of cells and cell line are good source. As far as primary cultures are concerned these are more relevant to the actual scenario as compared to the cell lines. So after standardization you can further validate the results on primary cultures and present it for publication. In my experience primary cultures are better and more reliable than cell lines.
  • Penelope Tsimbouri · University of Glasgow
    HI I do agree with everyone above.
    I tend to use cell lines for preliminary screening of the experiments I'm interested. Once everything is standardised then I move on to primary cells which are closer to the in vivo scenario.
    I recently worked with breast epithelial cells MCF7 line and MCF10A and also primary cells I isolated from primary breast tumours. So all the optimisation took place with the cell lines. When it came to the primary cells I split the original (positively identified breast epithelial cells) cultures into multiple flasks so I would have many of the same passage to optimise the experiment as well as to perform the actual experiment.
    I always make sure I use low passage of not only primary cells but also cell lines.
    This ensures reduced variation in your results. Also run cell line and primary cell work sets in parallel for confirmation that your assay works as before and also comparison.
    Make sure you always have frozen back ups to ensure continuation of the studies using the same lines.
    good luck
  • Seema Tripathy · Central Institute of Freshwater Aquaculture
    Cell lines are derived from primary cultured cells. When weculture the cells from tissues, organsand cells we call them as primary culture. But if this culture sustain for 120 days then it called a cell lie. And can be comercilized. We can buy cell lines but we have to initiate primary culture.
  • Ryan Dee · University College London
    I'd tend to agree with Stuart Jenkins in that if you are using cell lines, know what it is your working with. I saw a talk by Prof John Masters at UCL on cell line authentication. He is very much interested in the area and a campaigner for better practices with the use of cell lines. I'd recommend reading his work on the area if you're interested.
  • Saeid Abroun · Tarbiat Modares University
    All description is correct.
    Usually by primary cells or cell lines you will reach to biology data. By using another cell line you will able to confirm your finding. In next step, you are going to show the molecular mechanism of your new finding, but your primary cell may not be enough for several experiments, in this situation you will use cell lines, which able to passage and proliferate them.
    Yes, it has been possible that primary cell finds mutation during passage, and should be careful. In other hand, primary cell accept few passage and it will stop after several passage cause telomere will be short.
  • Maria Antal · University of Strasbourg
    I would add another point to the discussion: when using cell lines, it is important to know at each stage of differentiation cells were immortalized.
    As it has been said, the biological effects of some treatments can't be seen in cell lines because engeneering changed their biology. However, as they are easy to manipulate, it's a good start.
    Further, it is important to test first results in more physiological models, like primary cells, despite the difficulties in using them. Negative results in a cell line do not mean that it's really what happens in vivo. Therefore, I would continue in primary cells and even go further in organotypic cultures.
  • Karima Al-Salihi · University of Nottingham
    I do agree with all answers above.
    However , standardisation is very important in this case.
  • B.T. Altura · State University of New York Downstate Medical Center
    Results from our lab over many years indicates that passing primary cells over generations clearly changes the physiological ,biochemical and biophysical characteristics of rat, canine, and sub-human primate endothelial and VSM cells.
  • Karima Al-Salihi · University of Nottingham
    Please read the attach article

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