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Cell line immortalisation biosafety

Cell line immortalisation biosafety



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Cell lines are often immortalised by artificial expression of proteins which specifically cause the knockout of important cancer suppression genes or the activation of proto-oncogenes.

Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.

However, this would appear to fall under the purview of Biosafety Level 3 since the protein expression vectors are capable of causing cancer in the researcher when they infect the researcher. Cancer can certainly be considered a serious or lethal disease, and aerosolisation of the vectors, while rare, is not impossible (for example during a spill).

According to the biosafety definitions of Columbia University,

Agents: serious or lethal diseases transmissible via aerosols, e.g., M. tuberculosis, SARS. Recombinant DNA activities using genetic material from BSL-3 organisms or such organisms as host cells.

Why then, does the CDC classify cell line work, including work with viral production cells, as a BSL-2 activity requiring only BSL-2 containment levels, when the act of immortalising the cells would involve working with viral agents that can cause cancer?

Cells immortalized with viral agents such as SV-40, EBV adenovirus or HPV, as well as cells carrying viral genomic material also present potential hazards to laboratory workers. Tumorigenic human cells also are potential hazards as a result of self-inoculation.[… ]

Human and other primate cells should be handled using BSL-2 practices and containment. All work should be performed in a BSC, and all material decontaminated by autoclaving or disinfection before discarding. BSL-2 recommendations for personnel protective equipment such as laboratory coats, gloves and eye protection should be rigorously followed.

Are the transformation cells (eg the 293T viral cells containing the transformant viruses) considered Level 3 biosafety risk, and if not, why not?


Work with vectors expressing oncogenes or knocking down tumor suppressor genes (particularly if vectors are based on lentivirus) is often done under "BSL-2+" conditions. This essentially means that personal protective measures required for BSL-3 are used, but the specialized ventilation systems and so forth of BSL-3 physical laboratory space are not required. That would seem to address the investigator-safety issues raised in this question. See the complete Columbia policy as an example of the details and the risk-assessment process.

As noted already, once cultured cells have been infected with these vectors the risks to personnel are substantially diminished if not eliminated, so less stringent protections are then required.


Maybe, cells should be contained under BSL-2 and agents (viral particles etc) under stricter BSL-3? That is, after immortalization you can keep cells under lower security.

Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment.

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure

CDC Biosafety in Microbiological and Biomedical Laboratories, Laboratory Biosafety Level Criteria

As of HEK 293 cells, check this out:

Biosafety level 2 practices and containment facilities for all activities involving HEK-293 cell lines.

HEK 293 Cell Line Risk Assessment - IBC


Cell Lines: Types, Nomenclature, Selection and Maintenance (With Statistics)

The development and various other aspects of primary culture are described above. The term cell line refers to the propagation of culture after the first subculture.

In other words, once the primary culture is sub-cultured, it becomes a cell line. A given cell line contains several cell lineages of either similar or distinct phenotypes.

It is possible to select a particular cell lineage by cloning or physical cell separation or some other selection method. Such a cell line derived by selection or cloning is referred to as cell strain. Cell strains do not have infinite life, as they die after some divisions.

Types of Cell Lines:

Finite Cell Lines :

The cells in culture divide only a limited number of times, before their growth rate declines and they eventually die. The cell lines with limited culture life spans are referred to as finite cell lines. The cells normally divide 20 to 100 times (i.e. is 20-100 population doublings) before extinction. The actual number of doublings depends on the species, cell lineage differences, culture conditions etc. The human cells generally divide 50-100 times, while murine cells divide 30-50 times before dying.

Continuous Cell Lines :

A few cells in culture may acquire a different morphology and get altered. Such cells are capable of growing faster resulting in an independent culture. The progeny derived from these altered cells has unlimited life (unlike the cell strains from which they originated). They are designated as continuous cell lines.

The continuous cell lines are transformed, immortal and tumorigenic. The transformed cells for continuous cell lines may be obtained from normal primary cell cultures (or cells strains) by treating them with chemical carcinogens or by infecting with oncogenic viruses. In the Table. 36.1, the different properties of finite cell lines and continuous cell lines are compared.

The most commonly used terms while dealing with cell lines are explained below.

The divisor of the dilution ratio of a cell culture at subculture. For instance, when each subculture divided the culture to half, the split ratio is 1: 2.

It is the number of times that the culture has been sub-cultured.

It refers to the number of doublings that a cell population has undergone. It must be noted that the passage number and generation number are not the same, and they are totally different.

Nomenclature of Cell Lines:

It is a common practice to give codes or designations to cell lines for their identification. For instance, the code NHB 2-1 represents the cell line from normal human brain, followed by cell strain (or cell line number) 2 and clone number 1. The usual practice in a culture laboratory is to maintain a log book or computer database file for each of the cell lines.

While naming the cell lines, it is absolutely necessary to ensure that each cell line designation is unique so that there occurs no confusion when reports are given in literature. Further, at the time of publication, the-cell line should be prefixed with a code designating the laboratory from which it was obtained e.g. NCI for National Cancer Institute, Wl for Wistar Institute.

Commonly used cell lines:

There are thousands of cell lines developed from different laboratories world over. A selected list of some commonly used cell lines along with their origin, morphology and other characters are given in Table. 36.2.

Selection of Cell Lines:

Several factors need to be considered while selecting a cell line.

Some of them are briefly described:

In general, non-human cell lines have less risk of biohazards, hence preferred. However, species differences need to be taken into account while extrapolating the data to humans.

2. Finite or continuous cell lines:

Cultures with continuous cell lines are preferred as they grow faster, easy to clone and maintain, and produce higher yield. But it is doubtful whether the continuous cell lines express the right and appropriate functions of the cells. Therefore, some workers suggest the use of finite cell lines, although it is difficult.

3. Normal or transformed cells:

The transformed cells are preferred as they are immortalized and grow rapidly.

The ready availability of cell lines is also important. Sometimes, it may be necessary to develop a particular cell line in a laboratory.

5. Growth characteristics:

The following growth parameters need to be considered:

i. Population doubling time

ii. Ability to grow in suspension

iii. Saturation density (yield per flask)

The stability of cell line with particular reference to cloning, generation of adequate stock and storage are important.

7. Phenotypic expression:

It is important that the cell lines possess cells with the right phenotypic expression.

Maintenance of Cell Cultures:

For the routine and good maintenance of cell lines in culture (primary culture or subculture) the examination of cell morphology and the periodic change of medium are very important.

Cell Morphology:

The cells in the culture must be examined regularly to check the health status of the cells, the absence of contamination, and any other serious complications (toxins in medium, inadequate nutrients etc.).

Replacement of Medium:

Periodic change of the medium is required for the maintenance of cell lines in culture, whether the cells are proliferating or non-proliferating. For the proliferating cells, the medium need to be changed more frequently when compared to non-proliferating cells. The time interval between medium changes depends on the rate of cell growth and metabolism.

For instance, for rapidly growing transformed cells (e.g. HeLa), the medium needs to be changed twice a week, while for slowly growing non-transformed cells (e.g. IMR-90) the medium may be changed once a week. Further, for rapidly proliferating cells, the sub-culturing has to be done more frequently than for the slowly growing cells.

The following factors need to be considered for the replacement of the medium:

The cultures with high cell concentration utilize the nutrients in the medium faster than those with low concentration hence the medium is required to be changed more frequently for the former.

A fall in the pH of the medium is an indication for change of medium. Most of the cells can grow optimally at pH 7.0, and they almost stop growing when the pH falls to 6.5. A further drop in pH (between 6.5 and 6.0), the cells may lose their viability.

The rate of fall in pH is generally estimated for each cell line with a chosen medium. If the fall is less than 0.1 pH units per day, there is no harm even if the medium is not immediately changed. But when the fall is 0.4 pH units per day, medium should be changed immediately.

Embryonic cells, transformed cells and continuous cell lines grow rapidly and require more frequent sub-culturing and change of medium. This is in contrast to normal cells, which grow slowly.

4. Morphological changes:

Frequent examination of cell morphology is very important in culture techniques. Any deterioration in cell morphology may lead to an irreversible damage to cells. Change of the medium has to be done to completely avoid the risk of cell damage.


Immortalization and malignant transformation of eukaryotic cells

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.


Cell Immortalization

Several methods exist for immortalizing mammalian cells in culture conditions.
With years of experience in cell immortalization, our partner ABM have developed the most comprehensive cell immortalization product line comprising of recombinant lentiviral, retroviral (MMLV) and adenoviral viruses expressing EBV, HPV-16 E6/7 and SV40 T antigens, hTERT, p53 and RB siRNAs, and ras & myc mutants. All these tools will make your cell immortalization project simpler and easier than ever before.


Virus Induction: EBV, HPV E6/7 and SV40 T Antigen
One method is to use viral genes, such as EBV, HPV-16 E6/7 gene, and the Simian Virus 40 (SV40) T antigens, to induce immortalization. SV40 T antigens have been shown to be the simplest and most reliable agent for the transformation of many different cell types in culture and the mechanism of SV40 T antigens in cell immortalization is well studied. For the most part, viral genes achieve immortalization by inactivating the tumor suppressor genes (p53, Rb, and others) that can induce a replicative senescent state in cells. Recent studies have also shown that SV40 T antigen can induce Telomerase activity in the infected cells.

hTERT Expression
A more recently discovered approach to cell immortalization is through the expression of Telomerase Reverse Transcriptase protein (TERT), particularly for cells that are most affected by telomere length (e.g., human). This protein is inactive in most somatic cells, but when hTERT is exogenously expressed, the cells are able to maintain sufficient telomere lengths to avoid replicative senescence. Analysis of several telomerase-immortalized cell lines has verified that the cells maintain a stable genotype and retain critical phenotypic markers.
However, over-expression of hTERT in some cell types (especially in primary epithelial cells) fails to induce cell immortalization and it may induce cell death.

Inactivation of Tumor Suppression Genes
Recent research has found that co-expression of hTERT catalytic subunit with either p53 or RB siRNA can immortalize human primary ovarian epithelial cells, providing a more authentic normal cell model with well-defined genetic background (Yang G. et al. , Carcinogenesis 2007 Yang G. et al ., Oncogene 2007) . Likewise, overexpression of Ras or Myc T58A mutants have also been found to be able to immortalize some primary cell types (Sears R. et al ., Genes & Dev. 2000). The HOX genes (HOXB8, HOXA9, and HOXA10) are a family of homeodomain-containing transcription factors associated with malignancies such as acute myeloid leukemia and acute lymphoid leukemia. Accordingly, overexpression of the HOX genes can be used to immortalize various hematopoietic cells, including macrophages, hematopoietic progenitor cells, and myeloid progenitor cells.


Results

Comparison of Different Primary Cell Culture Methodologies

Initial trials comparing four different tissue culture methods (detailed in Materials and Methods) generated cell cultures of most tissue types with varying degrees of success. Generally, the methods using enzymatic digestion to break up the tissue (Methods 1 and 2) were more successful than the methods utilising physical disruption. (Methods 3 and 4). Method 2, trypsin treatment at 4°C overnight, was found to be the most effective and reliable in generating viable cell cultures across the majority of different tissue types. The comparatively long incubation time in trypsin allowed greater penetration and better digestion of the tissue as compared to Method 1, where trypsin treatment was at 37°C. The simplicity of Method 2 and its reproducibility led to the adoption of this method for our primary cell culture production. Contaminant-free cell cultures from intestine and skin were difficult to establish because of the obvious difficulty in obtaining tissues free from bacterial and fungal contamination.

Cell culture media was evaluated across the range of tissue type for optimal growth. Attempts to establish cell culture from tissues grown in Xten GO serum free medium was the least successful. The most successful cell culture medium across the majority of tissue types was found to be DMEM/F12-Hams. Supplementing media with bat serum as opposed to bovine calf serum appeared to make little difference to cell growth and so bovine calf serum was used for reasons of economy and convenience.

Preliminary Characterisation of Primary Cell Lines

During the establishment of the primary cell cultures, non-adherent cells were lost during changes of medium. Only cells that attached to the culture flask were maintained and propagated by passage. The initial primary cell cultures derived from most tissues were heterogeneous, with a variety of cell morphologies observable (Figure 1A). The growth rate of primary cell cultures varied considerably, with cells from the aorta, kidney and foetus growing to confluence quickly and requiring passage within 6 days. By contrast, muscle, brain and lymph nodes took up to 15 days to reach confluence. Varying cell morphologies were observed, ranging from predominantly fibroblastic-like cells observed in the majority of tissues to cuboidal cells in cultures generated from lung and kidney (Figure 1B). Neural cells with dendrites were observed in cultures generated from brain.

(A) Cells derived from a brain (left) and kidney (right) after 5 days in primary cell culture. (B) Cells derived from liver (left) and kidney (right) after 12 days in primary cell culture.

Once the primary cell lines were established, cells from all tissue types (Table 1) grew well and were able to be passaged a number of times. As the cell cultures were passaged further, the monolayers became more homogeneous in appearance and the variety of cell types in each culture decreased (Figure 1B). Typical of nearly all primary cell cultures, the growth of non-immortalised primary cell cultures diminished significantly for most tissue types after approximately 10 passages.

The identity of the bat cell lines established in this study was confirmed by two independent methods described in the Methods section. G-banding karyotyping demonstrated that the male P. alecto used for cell line development had 19 pairs of chromosomes, 18 pairs of autosomes plus one X and one Y (data not shown), with a similar morphology as that previously reported for a female P. alecto using R-banding [26]. A Pteropus-specific PCR was developed and validated using DNA extracted from the spleen of one female and one male P. alecto as well as DNA extracted from HeLa (human cervical cancer cell line), MDCK (Madin-Darby canine kidney), PK15 (pig), Vero (African Green monkey kidney), CHO (Chinese Hamster Ovary) and mouse heart tissue. While the predicted 454-bp fragment was obtained for P. alecto DNA, no PCR product was produced from the DNA samples of any of the other five mammalian species. Furthermore, sequencing of the P. alecto PCR product confirmed that it is highly conserved with the same region in the closely related P. vampyrus genome (data not shown).

Immortalisation and Cloning

Unlike rodent cells which are genetically relatively unstable [14], none of the bat primary cell lines established in this study appeared to have immortalised spontaneously. Therefore, two directed immortalisation strategies, i.e., the intracellular expression of SV40T or hTERT, were employed to transform the bat primary cell lines developed in this study. Both the SV40T and hTERT genes were introduced into bat cells using a retroviral vector system, which results in the stable integration of the introduced genes into the cellular chromosomal DNA [27]. Transformed cell lines are selected by using the hygromycin resistance marker encoded by the vector DNA. Expression of the SV40 large T antigen was confirmed by immunofluorescent antibody staining and Western blot, respectively (Figure 2). The expression of hTERT in transformed cells was also confirmed using the same methods (data not shown). Fifteen out of 20 primary cell lines were immortalised using the SV40 large T antigen approach and 12 using the hTERT approach (Table 1).

(A) Immunofluorescent staining of P. alecto spleen and kidney primary cells and cells transformed to detect expression of the SV40T antigens. (B) Western blot of untransformed P. alecto spleen and kidney primary cells (lanes 1 and 3, respectively) and transformed cells (lanes 2 and 4) to detect expression of the SV40 large T antigen.

Cloning of the newly immortalised cells was considered an essential step in the establishment of the cell lines. Cloning will necessarily reduce the heterogeneity of the cell types present in the cell line. If performed optimally, cloning will ensure that the cell line is derived from a single cell type. This is critical to the production of cell lines that have consistent, reproducible characteristics. We were able to isolate single cells and grow viable cultures from those cloned cells. At the time of writing, five cloned, immortalised cell lines have been established (Table 1). The Pteropus origin of all the clones has been confirmed by the Pteropus-specific PCR (described above). Stocks from all cloned cell lines have been frozen in liquid nitrogen and then subsequently resurrected. Only SV40T or hTERT treated cells were able to be cloned and passed more than 10 times, providing additional evidence of successful immortalisation.

Susceptibility to Infection by Nipah and Hendra Viruses

Variation in infectivity and viral protein production following high multiplicity infection with HeV or NiV was observed in the different primary cell lines. Although all primary P. alecto cell lines were successfully infected with both NiV and HeV, the infection efficiency was generally lower than that seen in the control infection in Vero cells (data not shown). In some primary cell cultures, only a small proportion of the cells produced a detectable level of viral protein expression 24 hours post infection (as detected by fluorescence, data not shwon). However, after 48 to 72 hours, all cell lines were producing greater quantities of viral proteins and for most primary cell lines every cell was infected. Only the primary heart cell line showed limited infection even after 72 hours (data not shown). The significance of this apparent difference is unclear. Similarly, in cloned immortalised cell lines, a difference was observed in infection kinetics in different lines. For example, NiV infected PaKiT02 cells (Figure 3A) produced detectable viral antigen levels comparable to that observed in Vero cells in almost all cells at 24 hours post infection, whereas the same infection in the PaKiH01 cells resulted in less than 25% of the cells producing detectable viral antigens at the same time point. However, at 48 hours, differences could no longer be seen.

(A) Comparison of infection kinetics of NiV in three different cell lines at 24 and 48 hours post infection. (B) Comparison of infection efficiency of P. alecto cloned cell lines for HeV and NiV. The images were taken 24 hours post infection. In both studies, cells were infected at high multiplicity of infection (MOI ≥100), fixed with 100% methanol and removed from the Biosafety Level-4 laboratory before being stained with HeV G protein-specific antibodies.

In general, there was no observable difference between HeV and NiV infectivity in any of the primary cell lines. However, distinctive difference in infection efficiency was observed in some cloned immortalised cells, with HeV having higher infection efficiency. As shown in Figure 3B, HeV appears to have a much higher infectivity than NiV in the foetus (PaFeT10) and brain (PaBrH04) clones immortalised with SV40T and hTERT, respectively.

Induction of Innate Immune Responses in Cloned Cell Lines

One of the major applications of the cell lines established in this study will be for the investigation of the innate immune responses to infection by viruses of both bat and non-bat origin. As a first step towards the characterisation of the innate immune competency of different P. alecto cell lines, the stimulation of type I interferon gene expression by poly I∶C was examined in selected SV40T cloned immortalised cell lines. The results presented in Figure 4 suggest that there is significant variation in the increase of type I interferon gene expression after poly I∶C treatment, from less than 10-fold increase in the PaFeT07 cells to more than 100-fold increase in the PaLuT02 cells. It is also interesting to note that the increase in IFN-β is greater than that for IFN-α in all the cell lines tested so far.

Results shown are of fold increase in IFN-α and IFN-β transcript levels (measured by real-time PCR) after treatment of P. alecto cloned and SV40 Large T antigen immortalised brain, kidney and foetal cells with 10 µg/ml poly I∶C over the basal level of IFN gene expression in mock treated cells. Error bars represent the standard deviation of the mean derived from duplicate samples.


Biosafety

Application

Primary cells or cell lines from any source other than human or primate

No aerosol management system (AMS) any sorter

Ex vivo human cells from prescreened group or human cell lines rated BSG 1 from ATCC

AMS, biosafety cabinet (BSC) recommended

Tissue from animals xenografted with noninfectious human cells

Primary human tissue retrieved from clinic tested for known pathogens HIV, Hep B, C, EBV, CMV

Genetically engineered human or animal cell lines with other than third-generation or older lentiviral or adenoviral gene transfer

Primary human cells from unscreened group or unknown patient history

Primary human cells from infectious patients

Biosafety Level 1 (BSL1): well-characterized agents not consistently known to cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment.

Biosafety Level 2 (BSL2): agents of moderate potential hazard to personnel and the environment.

Biosafety Level 3 (BSL3): indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by the inhalation route (applicable to clinical, diagnostic, teaching, research or production facilities).

Biosafety Level 4 (BSL4): dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease.

In 2009, the International Society for Advancement of Cytometry produced a guideline for cell-sorting core facilities to assist with understanding risks and biosafety terms. The guideline focused mainly on the most common types of cells and applications handled by cell-sorting facilities:


Cell Immortalization Reagents

Primary cells undergo a finite number of population doublings and reach a state called replicative senescence after limited cell divisions. GeneCopoeia&rsquos Cell Immortalization Reagents use recombinant lentivirus carrying genes, such as the simian virus 40 (SV40) T antigen, the human telomerase reverse transcriptase protein (hTERT), CDK4, HOXB, HOXA, cMyc, Bmi1 and HPV-16 E6/E7 to induce immortalization and significantly prolong primary cells' lifespan.

GeneCopoeia Lentifect&trade Lentiviral Particles are produced from a standardized protocol using purified plasmid DNA and the proprietary reagents EndoFectin&trade Lenti (for transfection) and TiterBoost&trade solution. The protocol uses a third-generation self-inactivating packaging system meeting BioSafety Level 2 requirements.

The Lentifect&trade particles include CMV or EF1&alpha promoter for efficient expression of the immortalization inducer gene in target cells with puromycin or hygromycin resistance marker for selection of stably transduced cells. The immortalization lentivirus particles without antibiotic selection marker are also available.

Provided as 4 vials of 25 µl of lentiviral particles with titers of

1 x 10 8 TU/ml. Lentifect&trade particles are shipped on dry ice and must be stored at &ndash80°C immediately upon receipt. Avoid repeated freeze-thaw cycles as this will reduce titers.

The lentiviral expression construct was validated by full-length sequencing, restriction enzyme digestion and PCR-size validation using gene-specific and vector-specific primers. The product is confirmed free of bacteria, fungi and common Mycoplasma contamination.


ASSESSMENT OF AEROSOL CONTAINMENT

The classic method for assessment of aerosol containment on deflected-droplet cell sorters using aerosolized bacteriophage and a detection system of bacterial lawns has been described in several publications ( 1 , 12 , 37 , 39 ). The T4 bacteriophage method for assessment of containment can also be combined with active air sampling for testing room air ( 1 , 38 , 39 , 45 ). The T4 settle plate method and the active air sampling method are described in detail in Appendix 1. Tagging aerosol droplets with bacteriophages is an established technique that, provided the titer of the bacteriophage is sufficiently high, insures that all droplets generated during the test sort contain T4. Because Merrill has established that a single phage is sufficient to generate one plaque ( 12 ) the assay provides high sensitivity. Furthermore, by counting plaques, the readout of containment results is straightforward. This method is an overnight procedure requiring intermediate knowledge of microbiological techniques and depends on the performance of biological materials.

Recently, a novel assay for measuring the efficiency of aerosol containment has been described ( 42 , 43 ). The method uses a suspension of highly-fluorescent melamine copolymer resin particles, which simulate a biological sample during the test sort. Aerosol containment is measured by placing microscope slides around the instrument where aerosols are produced and could escape. The slides are examined for the presence of particles under a fluorescent microscope. Perfetto et al. ( 40 , 44 ) have increased the assay's sensitivity and reproducibility by using a viable microbial particle sampler. This device draws room air onto a microscope slide and concentrates the collected resin particles onto the areas on the slide located directly underneath the intake ports. This technique is described in Appendix 1 and is suitable to be performed immediately before a potentially biohazardous sort however, for this practice a fluorescent microscope has to be readily accessible. The highly fluorescent particles can be easily detected, but careful handling of the microscope slides, and the air sampler are important to avoid false positives. Diligent scanning of the entire slide is required to reliably detect escape of a single particle.

Before sorting any unfixed and potentially bio-hazardous specimens on a given instrument, it is imperative to validate that aerosols are contained during the regular sorting process and during instrument failure modes [Appendix 1]. If aerosols are detected outside of containment, then the cell sorter must be modified such that no aerosols are detectable. Contacting the instrument manufacturer for instructions or dispatching a service engineer will be necessary before making any instrument modifications. Testing must also be done whenever changes are made to the cell sorter that may affect escape of aerosols, e.g., installation of a new drive head or flow cell, replacement of the sort chamber door, or alterations in the aerosol management system.

For instruments that are placed into biological safety cabinets, it is imperative that laboratories validate initially at installation the efficiency of aerosol containment of the cabinet before any potentially bio-hazardous sorting experiments are performed. Frequent retesting and monitoring proper functioning of the cabinet is mandatory.

Since every live infectious sort has the potential to create infectious aerosols, verification of aerosol containment should be performed as often as possible. It is strongly recommended to perform testing prior to every infectious cell sort and maintain a record of the results. This practice will assure validation of the aerosol management system to contain aerosols containing potentially infectious pathogens.


Acquiring cell lines

You may establish your own culture from primary cells, or you may choose to buy established cell cultures from commercial or non-profit suppliers (i.e., cell banks). Reputable suppliers provide high quality cell lines that are carefully tested for their integrity and to ensure that the culture is free from contaminants. We advise against borrowing cultures from other laboratories because they carry a high risk of cell culture contamination. Regardless of their source, make sure that all new cell lines are tested for mycoplasma contamination before you begin to use them.

We offer a variety of primary cultures and established cell lines, reagents, media, sera, and growth factors for your cell culture experiments.


Author information

Present address: Genzyme Corporation, Cambridge, 02139, Massachusetts, MA, USA

Present address: Biogen, Inc., Cambridge, 02142, Massachusetts, MA, USA

Affiliations

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, 02142, Massachusetts, MA, USA

Ante S Lundberg, Sheila A Stewart, Brian Elenbaas, Kimberly A Hartwell, Mary W Brooks, Robert A Weinberg & William C Hahn

Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, NC, USA

Scott H Randell, John C Olsen & Scott W Miller

Department of Adult Oncology, and Department of Medicine, Dana-Farber Cancer Institute, Brigham and Women's Hospital, 44 Binney St., Boston, 02115, Massachusetts, MA, USA

Ante S Lundberg & William C Hahn

Department of Medicine, Harvard Medical School, Boston, 02115, Massachusetts, MA, USA

Ante S Lundberg & William C Hahn

Department of Pathology, Children's Hospital and Harvard Medical School, Boston, 02115, Massachusetts, MA, USA

Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, MA, USA


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