All Categories
Cell Culture

#### Why are my cells not attaching or proliferating after thawing?

Subscribe to our knowledge base and be notified about new articles.
Cell Culture – Introduction

Video Summary

Cells, the fundamental unit of life, are an incredibly useful tool in biological research. They serve as an imperative model system for understanding physiological processes and screening of toxic or therapeutic compounds for use in medical treatments. In addition, cells play a major role in functional enzyme, growth factor and vaccine production, along with a multitude of other uses. In order to accomplish aforementioned practices, cells must first be cultured in an environment outside from the organism they originate. This is the process of cell culturing. To elaborate, cell culturing involves maintaining cells of multi-cellular organisms outside of their original body under precise conditions (1).

Importance of Cell Culture

In vaccine research:

Cell culture has a diverse range of uses as cultured cells are used by cell biologists, biomaterials scientists, clinicians and regulatory authorities, among others (2). One of the most important uses of cell culture is in the research and production of vaccines. The ability to grow large amounts of virus in cell culture eventually led to the creation of the polio vaccine, and cells are still used today on a large scale to produce vaccines for many other diseases. Early in the 1930s and 40s, researchers had to use live animals to grow poliovirus, but with the advent of cell culturing, they were able to achieve much greater control over virus production and on a much larger scale (3). This allowed researchers to study them more closely and eventually develop vaccines and various treatments.

In protein therapeutics:

Another important use of cell lines is to express different types of proteins in mammalian cells (2). Initially, E. coli was the primary organism used for producing proteins, but with the need to create properly folded proteins with the proper post translational modifications, the focus shifted to using eukaryotes instead (4). Starting from the 1970s and 80s, proteins like interferon and antibodies have been successfully created via cell culture. Various cytokines and growth factors can be acquired from cultures, and looking at the structure and activity of these proteins helps us understand their role in the organism’s body as well. Even observing the protein grown in different cells and conditions may help us determine how the protein’s environment affects its activity.

In cancer research:

Cancer is one of the leading causes of death around the world and millions of dollars are being used for cancer research to find treatments and cures. Cell culture is crucial here as well, as normal cells can be transformed into cancer cells by methods including radiation, chemicals and viruses (5). These cells can then be used to study cancer more closely and to test potential new treatments.

Categories of cell culture: Primary Cells vs. Cell Lines

The 2 main types of cells used in laboratories are primary cells and established cell lines.

Primary cells, also known as finite cells, are cells that are directly prepared from an organism’s tissues (6). If grown under the right conditions primary cells will grow and proliferate, but they are only able to do so a finite number of times (7). This number is known as the Hayflick limit (7). The Hayflick limit is related to the telomere length at the end of the cell’s DNA. As cells undergo each cell division, a small segment of the telomere is lost after each time DNA is duplicated. This process eventually leads to a stage of senescence, where the cells can no longer divide. Continuous cell lines, on the other hand, are able to escape the normal constraints of the cell cycle and grow indefinitely, making them extremely useful for long-term research (8). Whereas primary cells are obtained directly from donor tissues, cell lines can be derived from clinical tumors, or created from transforming primary cells with viral oncogenes or chemical treatments.

 We offer a wide collection of primary cells and immortalized cells from human, mouse and rat.
Cell Cycle and Immortalization

While primary cells are known for their virtue of retaining most of the characteristics of the original tissue from which they are derived from, they are difficult to maintain in culture and can have batch-to-batch variation. Immortalized cells offer a solution to this setback and present a constant supply of research material.

In order to understand how cells are immortalized, it is first important to understand the mammalian cell cycle. There are 5 phases in the mammalian cell cycle: three gap phases, G0, G1 and G2, a synthesis phase and a mitosis phase (Figure 1) (9). These 5 phases can be clumped together into 2 major parts: interphase, which includes the gap phases and synthesis, and mitosis (9). When a cell is in G0, it is simply in a quiescent state and is not preparing to divide. The binding of mitogens, like growth factors, will cause the cells to leave G0 and enter G1, thereby preparing to divide. Mitogens are substances that induce cells to undergo mitosis (10). During G1, S, and G2, the cell is making RNA, various proteins, and duplicating DNA in order to prepare for division, i.e. mitosis (9).

Figure 1 – The Cell Cycle

There are several checkpoints in between phases in the cell cycle that ensure that the cell meets the appropriate requirements in order to move on to the next stage. The first checkpoint is at the end of G1. Known as the restriction point, it allows cells to commit to cell division. The second is in S-phase and is there to check the quality of the replicated DNA and to determine if any DNA repair mechanisms need to be activated. The third checkpoint is after G2, and its purpose is, again, to ensure that DNA has been replicated completely and is undamaged. Finally, there are several checkpoints within mitosis to ensure that the cell is in a proper position to complete cell division, i.e. cytokinesis (9).

There are various molecules that are in charge of controlling the stages of the cell cycle, molecules like cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (9). Generally, cyclins bind to Cdks in order to form complexes that are then able to move the cell from one part of the cycle into the next (9). Cyclins are created and destroyed throughout the cell cycle and their levels can help to determine approximately what stage of the cycle the cell is in (9). Cdk inhibitors, as their name suggests, can bind to these cyclin-Cdk complexes and prevent them from initiating the next stage in the cycle (9).

Another protein, the tumour suppressor protein p53, is also vital, both to the cell cycle and immortalizing cells. It comes into play at the checkpoints after G1 and G2; if there is DNA damage, it will induce transcription of the Cdk inhibitor p21, which will prevent the activation of G1 cyclin-Cdk complexes (9). Inhibiting p53 will allow the cell cycle to progress and continue proliferating, and can result in cell immortalization. Some ways to do this are by introducing the E6 and E7 oncogenes or the E1a and E1b proteins, among other possible methods (10)(12).

The E6 and E7 oncogones from the Human Papillomavirus can degrade the tumour suppressors p53 and pRb, which, as mentioned previously, play crucial roles in preventing the progression of the cell cycle at the G1 and G2 checkpoints (13)(14). E6 can also induce the expression of the hTERT gene, which codes for a telomerase that can prevent the degradation of telomeres at the ends of DNA (14). The E1 proteins from some adenoviruses are also able to bind p53 and has effects similar to those of the E6 and E7 oncogenes (15).

There are many other ways to immortalize cells. One way is to introduce a catalytic version of telomerase into cells to prevent their telomeres from shortening (6)(10)(16). Telomeres shorten over time and it is thought that it is this shortening that leads to aging and cell death (16). Another way is to alter cell cycle checkpoints like inactivating p53 and pRb, both major controllers of the cell cycle (10) (Figure 2).

Figure 2 – p53 and pRb in cell cycle

It is important to note that not all immortalized cells will retain the same characteristics as the primary cells and often times characterization experiments are performed on the newly established cell lines to determine their usefulness in research settings.

 We offer immortalizing reagents, such as recombinant lentivirus encoding HPV E6/E7, hTERT, viral oncogenes SV40, or siRNA targeting p53 and Rb here.
Cell Growth Environment

There are two main growth conditions in cell culture: as monolayers (i.e. adherent culture) or free-floating in culture media (i.e. suspension culture) (17). Adherent cells adhere to the culture vessel with the use of an extracellular matrix (ECM). ECMs are generally derived from tissues of organs that are immobile and embedded in a network of connective tissue (17). Suspension cells are able to grow while suspended in their liquid medium. Suspension cells are usually derived from cells in the blood system, like lymphocytes (17).

Cell Culture Media

Different cell lines generally require specific types of media (7). All media will include amino acids, inorganic salts and vitamins (7). Usually, fetal bovine serum is added to media prior to use as it provides vital macromolecules, growth factors and immune molecules (7). As there is a risk of bacterial and fungal contamination in cell culture, very commonly Mycoplasma contamination, antibiotics like Penicillin and Streptomycin are often added to prevent it (7). There are many different kinds of media available in the market today, but some of the more commonly used ones are DMEM, RPMI and F12.

Media Buffering System

The pH of cell culturing media is also important as drifting outside of the cells desired pH may have harmful effects on the cells (18). To ensure that the proper pH is maintained, a buffering system such as sodium bicarbonate is added to media (18). The sodium bicarbonate in the media maintains a pH between 7.2 and 7.4 with the gaseous CO2, which is ideal for most cell lines (18). Culturing using the bicarbonate buffering system requires the presence of 5-10% CO2 in the air and this is commonly achieved with the cell culture incubator. Alternatively, a chemical buffering system involving the use of zwitterion ions (i.e. HEPES) can also be used. A controlled gaseous atmosphere is not required with HEPES buffered cultures. Media will also contain pH indicators like phenol red that not only provide the media with a certain color, but also allow scientists to qualitatively determine the approximate pH of a culture’s media (18).

Defined Media

As mentioned previously, media usually has several generic components but when adding ingredients like fetal bovine serum, it is hard to determine the serum’s exact composition. For example, there is the possibility of fetal bovine serum containing prions if the serum is harvested from a cow with Bovine Spongiform Encephalopathy (BSE) (19). Additionally, different batches of FBS will likely have slightly different compositions as well, and this could have various effects on the cell culture. To resolve possible issues like this, defined media were created. In defined media, all the components are properly identified and are added in exact concentrations (20); these components may include growth factors and lipoproteins for example.

Temperature

Cells can differ in their preference of the conditions in their external environment. For example, some cells prefer growing in 37°C, while others prefer 33°C. These conditions must be monitored strictly as diverging from these conditions can greatly reduce cell proliferation and differentiation (22). Most mammalian cells are maintained at 37˚C for optimal growth, while cells derived from cold-blooded animals tolerate a wider temperature range (i.e. 15˚C to 26˚C).

Culture Vessels

There are many different types of containers that can be used to culture cells. These containers vary in size, shape, coating, and the presence or absence of a lid. Sizes can range from the small 96-well plates to much larger T175 flasks (Figure 3). Several different types of coatings that are used are collagen, gelatin, and fibronectin. The coatings are all types of extracellular matrix, which serve to mimic conditions present in the cell’s natural environment and can help them grow much better (23). Depending on the experimental requirements, cells can be moved freely between the different culture vessels. Scale-up experiments are possible with roller bottles, spin cultures and CellStacks, among many other options.

Figure 3 – Standard cell culture flasks

Cell Culture Laboratory

When culturing cells, sterile lab techniques are absolutely essential. Bacterial infections, like Mycoplasma, and fungal infections in cell culture can be very problematic to identify and eliminate (7). As such, it is imperative that all cell culture work is done in a sterile environment with proper aseptic technique; there are also several things to take into consideration.

Laminar Flow Hood

A laminar flow machine is always used to do cell culture work, and is thoroughly sprayed down with 70% ethanol before and after use (7). A laminar flow machine maintains a steady, clean airflow and prevents outside contaminants from entering the culture (Figure 4).These machines take in air from the outside environment, send it through their HEPA-filtering system, and exhaust the clean air across the working surface to create a sterile work environment (24). Many hoods generally also have UV lights that can be turned on after use to sterilize the interior, by killing any pathogens that may be present. When items like media and tubes are being placed in the hood, they must also be sprayed down with 70% ethanol (7) . When media is being used, the lid must be kept open for as short a time as possible and the lid opening should face the work surface in order to prevent contaminants from entering it (7). Finally, gloves must always be worn when doing cell culture work and should be cleaned regularly with 70% ethanol (7).

Figure 4 – Laminar flow hood

Biological Contamination

Contamination of cultures are the most commonly encountered problem in cell culture. Biological contaminations include bacterial, fungal, viral, as well as cross-contamination by other cell lines. Sometimes, contamination can be clearly seen in cell culture. For example, if there is bacterial contamination, the media will seem cloudy and when viewed under the microscope, millions of tiny particles much smaller than human cells can be seen. At other times, it is not so obvious but it is possible to test cultures for various contaminants. For example, one way to detect Mycoplasma contamination is with PCR. DNA can be extracted from the cells in a culture, primers specific to sequences in Mycoplasma species can be made, and the DNA can be run in a PCR reaction with the primers to detect the presence of Mycoplasma DNA (25). There are many kits in the market that can be used to detect Mycoplasma. Viral infection is another contamination that is difficult to detect, and often immunostaining or ELISA assay is required to detect viral associated-proteins in cultures.

 Routine checks for mycoplasma contamination is recommended for cell culture. Learn how to rapidly detect contamination using our PCR detection kit.

Cross-contamination

When culturing cells, it is extremely important to ensure that the cells being cultured have not been contaminated with a different cell line. Cell lines can be overgrown and replaced by another fast-growing cells inadvertently introduced into the original culture. The consequence of using a misidentified cell line is making false scientific conclusions.

One way to authenticate cell lines is by using a method called Short Tandem Repeat (STR) profiling. However, it must be noted that it can only be used with human cells (26). The DNA genome in human cells have hypervariable regions that have multiple repeated sequences, called variable number tandem repeat (VNTR) units (27). These repeat sequences are generally 1-6 base pairs long, and the number of repeats and the actual sequence are unique to each individual (27). These sequences are amplified using PCR and are then used to create a standard profile for each cell line (27). The standard profiles are subsequently used as a reference that researchers can compare their cell lines to in order to ensure the correct identity (27).

 Many scientific journals and funding agencies now require evidence of cell line authentication by STR profiling as a requisite for publishing or obtaining funding. Get your STR profiling service here.
Cell Culture Techniques

There are many different techniques that are used in cell culturing. Some are used to directly culture or freeze cells, others are used to authenticate cell lines and check for cross-contamination between cell lines. Below are some brief summaries of some of the more common techniques.

Subculturing

After they are grown in culture for a few days, cells can become too crowded for their container and this can be detrimental to their growth, generally leading to cell death if left for long periods of time as they will use up the nutrients over time (7). A common solution to this is to subculture the cells into another container. What this involves is simply taking a portion of the cells from one container and moving them to a new container with fresh media, thus providing more space and nutrients for both portions of cells to grow (7).

If the cells are suspension cells, they can simply be transferred into a conical tube, spun down in a centrifuge, and then re-suspended in fresh media. This suspension can be divided into 2 or more portions and added to new containers. However, if the cells are adherent cells, they must first be detached from their container (7). Trypsin is a common proteolytic enzyme which breaks down proteins that help cells adhere to culture vessels. Therefore, trypsinization, the process of cell dissociation using trypsin, is used to passage cells into a new vessel (Figure 5) (7).

Figure 5 – Basic Subculturing Procedure

Other dissociation enzymes such as dispase or collagenase, both of which are gentler than trypsin, can also be used for digestion. Prolonged exposure to trypsin can damage the proteins on the cell’s surface and thereby affecting subsequent cell attachment and cell functioning. Therefore, trypsin must be diluted and inactivated after most cells are detached from the culture vessel. Inactivation is commonly achieved by the addition of serum and divalent cations calcium and magnesium (often in the form of serum-containing media). In cases where serum-free conditions are used to culture cells, soybean trypsin inhibitor can be added to prevent further tryptic activity. After the cells are freed from the culture vessels, the cells can then be transferred to a conical tube, spun down, and then portioned into new containers using fresh media (7).

 We offer Trypsin-EDTA for various cell culture applications, including detachment of adherent cells, enzymatic dissociation of tissues into single cell suspensions and dissociation of ES cell colonies.

Seeding Density

One important factor to keep in mind when subculturing cells is the seeding density, which is the number of cells being added into a culture vessel. Some cell lines prefer high seeding densities while others are able to proliferate well even under low densities. Some will even change their characteristics depending on the seeding density. For example, granulosa cells in culture will secrete estradiol at lower densities but will gradually transition to secreting more progesterone as the density increases (28). If the cells are known to grow fast or if the cells will be cultured for a longer period of time, larger culture vessels should be used.

Propagation, Population Doubling and Passage Number

Another important factor to keep in mind is the phase of proliferation that the cells are in. There are four phases: lag, log, stationary and decline (29). During the lag phase, the number of cells is not increasing and the cells are simply acquiring enough resources to prepare for proliferation (29). During the log phase, the cells begin to proliferate and do so faster and faster over time. At the stationary phase, the rate of proliferation plateaus off and maintains a constant rate, due to the lack of nutrients and the presence of toxic metabolic waste products (29). Finally, when the cells reach the decline phase they begin to die off due to a lack of nutrients, altered pH conditions and the accumulation of toxic waste products (29). Cells should be seeded when they are in the lag phase so they can enter the log phase whilst in their new media (30). Generally they are frozen while in the log phase (25).

Population doubling in cell culture is simply the time it takes for a cell population to double. Another related term is the population doubling level, which is used to describe the total number of times the cells in a culture have doubled since they were first grown in vitro (31). A common formula used to calculate the population doubling level is (32):

$${Population \ Doubling \ Level} = {Log^{10}({N \over N_0}) \times 3.33}$$

Where N is the number of cells in the culture vessel at the end of a certain time interval, and No is the original number of cells plated in the vessel.

Population doubling and passage number are often mixed up or thought to mean the same thing. The passage number actually describes the number of times that a culture has been subcultured (32). In contrasts with the population doubling level in that the specific number of cells involved is not relevant. It simply gives a general indication of how old the cells may be and how suitable they would be for various assays (23).

Cryopreservation

Often times, cells are frozen down (i.e. cryopreserved) for various reasons, for example, generating cell stocks to make sure that the culture is not lost due to unexpected equipment failure or biological contaminations. Keeping cells in storage can also eliminate the time, energy and materials that may otherwise be required to maintain cultures not in use. Most importantly, cryopreservation can prevent finite cells from reaching senesce and minimize risks of phenotypic drift due to genetic instability in long term cultures.

i. Freezing Cells

The first step in freezing cells is to collect a cell pellet; depending on whether the cells are adherent cells or suspension cells, there are slightly different protocols. If they are suspension cells, the suspension can simply be collected into a conical tube, spun down in a centrifuge, and collected as a pellet after the media has been removed. If they are adherent cells, the media must be removed and trypsin added to free the cells from the container’s surface, much like in subculturing. After adding serum-containing media to neutralize trypsin, the cells can again be spun down in a tube to collect a pellet (32).

Once a pellet has been collected, the final step for both adherent and suspension cells is to re-suspend the cells in cryopreservation media. As the cell suspension cools down, the cells will lose water and the concentration of solutes will increase. In addition, ice crystals will form and damage the cells. To prevent loss in viability, cryoprotectant agents such as dimethyl sulphoxide (DMSO) or glycerol are included in the cryopreservation media (34) in order to protect the cells from potential damage caused by ice crystals and osmotic effects (23). Serum is commonly added as part of the mixture as well to protect the cells from intracellular ice crystals (Figure 6) (23).

Figure 6 – Thawing, subculturing and freezing prcedures at a glance: Thawing, subculturing and freezing must be done as quickly as possible as excess time outside of their preferred media can result in cell damage.e

A controlled and slow cooling rate, typically at -1˚C per minute, is critical in cryopreservation as it allows water to slowly escape from the cells through osmosis, which in term will minimize ice crystal formation inside the cells. Long term storage of cells require temperatures below -130˚C, usually in a specialized electric freezer or liquid nitrogen freezers. Cells can be stored and recovered decades after it is frozen down.

 We offer both standard and serum-free ready-to-use cryopreservation media that will improve cell recovery after thawing.

ii. Thawing Cells

When thawing the cells, the frozen tube of cells is warmed quickly in warm water, rinsed with medium and serum in order to remove the toxic DMSO, and then added into culture containers once suspended in the appropriate media (32).

Terminology

Cell culturing, much like many other parts of biological research, has its own set of unique vocabulary. Below are some common terms used in cell culturing; familiarity with these will help in understanding any literature and techniques about cell culture.

Adherent cells: Cells that are able to adhere to their culture vessel with the use of an extra-cellular matrix (ECM).

Attachment efficiency: The percentage of cells that actually adhere to the culture vessel’s surface, within a specified period of time, after inoculation.

Aseptic technique: Procedures that are used to prevent contamination of cell, tissue and organ cultures with fungi, bacteria, viruses, mycoplasma and other microorganisms.

Cell culture: The maintenance of cells in vitro outside of their original body.

Cell line: The result of the first subculture of a primary cell line.

Cell strain: Derived from a primary culture or cell line, a cell strain has cells of a specific type with unique properties or markers.

Cryopreservation: The storage of cells, tissues, embryos or seeds in extremely cold temperatures, usually below -130°C.

Epithelial cells: Cells that grow very close together in order to form continuous mosaic-like sheets. When other cells adhere to each other, they are referred to as “epithelial-like cells”.

Fibroblasts: Cells that have a spindle or irregular shape, that a responsible for the formation of fibers. It is often hard to distinguish fibroblasts from others types of cells in cultures, leading to the term “fibroblast-like cells”.

Finite cell culture: A culture that is capable of only a limited number of subculturing rounds, after which the cells cease to proliferate.

Immortalization: Attaining a cell culture that is able to proliferate continuously.

Passage: The transfer of cells from one culture vessel to another. A more specific term is subculturing where the cells are first subdivided before being transferred into multiple cell culture vessels. A passage number will refer specifically to how many times a cell line has been subcultured.

Primary culture: A culture started from cells, tissues or organs that are taken directly from the organism.

Suspension culture: A type of culture where cells multiply while suspended in their liquid medium.

References
• Cell Culture. http://www.britannica.com/science/cell-culture. Accessed July 6, 2015.
• Marquis CP. Mammalian Cell Culture. Vol 1.
• N/A. Early Tissue and Cell Culture in Vaccine Development. 2014. http://www.historyofvaccines.org/content/articles/early-tissue-and-cell-culture-vaccine-development. Accessed July 6, 2015.
• Moreira AR. Protein Expression and Cell Culture. 2007;(October).
• Roy, S. Kumar, V., Barman, D., Kumar, A. Paul, L., Datta M. Cell Culture & Its Application. http://aquafind.com/articles/Cell_Culture.php.
• Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 4th Edition.; 2002.
• Donaldson C. Cell culture. Br J Hosp Med. 2015;76(1):C2-C5.
• Stacey G, Macdonald C. Chapter 3 Immortalisation of Primary Cells. Cell. 2001:231-246.
• Humphrey T, Brooks G. Cell Cycle Control.; 2005.
• Offermanns S. Mitogen. In: Encyclopedia of Molecular Pharmacology. Vol ; 2008:776.
• Maqsood MI, Matin MM, Bahrami AR, Ghasroldasht MM. Immortality of cell lines: Challenges and advantages of establishment. Cell Biol Int. 2013;37(10):1038-1045. doi:10.1002/cbin.10137.
• Jha KK, Banga S, Palejwala V, Ozer HL. MINIREVIEW SV40-Mediated Immortalization. 1998;7:1-7.
• Piltti K, Kerosuo L, Hakanen J, et al. E6/E7 oncogenes increase and tumor suppressors decrease the proportion of self-renewing neural progenitor cells. Oncogene. 2006;25(35):4880-4889. doi:10.1038/sj.onc.1209492.
• Münger K, Baldwin A, Edwards KM, et al. Mechanisms of Human MINIREVIEW Mechanisms of Human Papillomavirus-Induced Oncogenesis. J Virol. 2004;78(21):11451-11460. doi:10.1128/JVI.78.21.11451.
• Moran E. Interaction of adenoviral proteins with pRB and p53. FASEB J. 1993;7(10):880-885.
• Hayflick L. A Brief History of the Mortality and Immortality of Cultured Cells. Keio J Med. 1998;47(3):174-182. doi:10.2302/kjm.47.174.
• N/A. 3 Types of cell cultures. https://moodle.kent.ac.uk/external/mod/book/view.php?id=2604&chapterid=160. Accessed July 6, 2015.
• Yang Z, Xiong H. Culture Conditions and Types of Growth Media for Mammalian Cells. Biomed Tissue Cult. 2012:3-18. doi:10.5772/52301.
• Epstein D, Monsell R, Horwitz J, Lenk S, Ozturk S, Marsh C. Chemically defined media compositions. 2006. http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&d=PALL&RefSrch=yes&Query=PN%2F7598083.
• Jayme DW, Smith SR. Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture. Cytotechnology. 2000;33(1-3):27-36. doi:10.1023/A:1008133717035.
• Todar K. Nutrition and Growth of Bacteria (page 3). http://textbookofbacteriology.net/nutgro_3.html.
• Harris I. Animal Cell Culture. Vol 21. 1993. doi:10.1016/0307-4412(93)90121-F.
• Clynes M. Animal Cell Culture Techniques.; 1998.
• This I, Cabinets BS, Levels B, et al. Appendix A – Primary Containment for Biohazards : Selection , Installation and Use of Biological Safety Cabinets. Personnel.:290-325.
• Uphoff CC, Drexler HG. Detecting Mycoplasma Contamination in Cell Cultures by Polymerase Chain Reaction. Cancer Cell Cult Methods Mol Biol. 2011;731:93-103.
• Barallon R, Bauer SR, Butler J, et al. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues. In Vitro Cell Dev Biol Anim. 2010;46(9):727-732. doi:10.1007/s11626-010-9333-z.
• Reid Y, Storts D, Riss T, Minor L. Authentication of Human Cell Lines by STR DNA Profiling Analysis. In: Sittampalam GS, Couseens NP, Nelson H, et al., eds. Assay Guidance Manual. Vol ; 2013.
• Portela VM, Zamberlam G, Price C a. Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells. Fertil Steril. 2010;93(6):2050-2055. doi:10.1016/j.fertnstert.2009.01.151.
• Nagabhushanam R. Fouling Organisms of the Indian Ocean.; 1997.
• Guidelines for Maintaining Cultured Cells. http://www.lifetechnologies.com/ca/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/maintaining-cultured-cells.html. Accessed July 11, 2015.
• King RC, Mulligan PK, Stansfield WD. A Dictionary of Genetics.; 2013.
• Mather JP, Roberts PE. Introduction to Cell and Tissue Culture: Theory and Technique.; 1998.
• Pollard JW, Walker JM, eds. Basic Cell Culture Protocols.; 1997.
• Walker MJ, Rapley R, eds. Molecular Biomethods Handbook.; 2008.
• Schaeffer WI. Terminology Associated with Cell, Tissue and Organ Culture, Molecular Biology and Molecular Genetics. Vitr Cell Dev Biol. 1990;26(1):97-101.
• Fedoroff S. Proposed Usage of Animal Tissue Culture Terms. Cytogenet Genome Res. 1967;6(3-4):161-167. doi:10.1159/000129938.

Top