In a human, what non-germline cells have the highest/lowest mass?

In a human, what non-germline cells have the highest/lowest mass?

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I'm just curious which cells are largest/smallest in the human body other than sperm/ova.

Large cells: adipocytes, the cells that store fat are possible the largest in humans, because after puberty they rarely divide and just enlarge if a person gains weight. A typical adipocyte is about 0.1mm in diameter, but may be a lot larger. Another good candidate is a megakaryocyte, which is a gigantic (up to 0.1mm), cell with multiple copies of its genome producing blood platelets. Depending on your definition of a single cell, also myocytes may be considered large cells, because during development several cells fuse to form one muscular fibre. Additionally, myocytes gain weight if a person excersises and strengthens muscles.

Small cells are more common, and it is difficult to point to some consistently smallest cells. The smallest kind-of-cells are definately platelets and erythrocytes (red blood cells - they are anucleated, thus may not be considered "real" cells). Generally, the part that determines the minimum size of the cell is the nucleus, so any cells, that are more-or-less metabolically inactive may be just a tiny bit larger than the size of their nucleuses. These may be for example polar cells, the tiny cells, that carry the excess of genome during meiosis in women and die soon afterwards.

If you are interested in all kinds of strange cells inside yourself, you might start by browsing this beautiful, though longish list.

Stem Cell Research

3.2.3 Fetal Stem Cells

Fetal stem cells were first isolated and cultured by John Gearhart and his team at the Johns Hopkins University School of Medicine in 1998 ( Shamblott et al., 1998 ). These cells known as primordial germ cells are the precursors of eggs and sperms and were isolated from the gonadal ridges and mesenteries of 5–9-week fetuses obtained by therapeutic abortion. Embryonic germ (EG) cells isolated from them are found to be pluripotent. Problems associated with isolation of these stem cells are (1) they can be obtained only from 8- to 9-week-old fetuses and (2) EG cells have limited proliferation capacity.


Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein–protein interaction or membrane–membrane apposition. At endoplasmic reticulum–mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum–mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.

Proximity labeling (PL) has been shown to be a valuable tool for studying protein localization and interactions in living cells (1 ⇓ –3). In PL, a promiscuous enzyme such as APEX (4, 5), BioID (6), or TurboID (7) is genetically targeted to an organelle or protein complex of interest. Addition of a biotin-derived small-molecule substrate then initiates biotinylation of endogenous proteins within a few nanometers of the promiscuous enzyme, via a diffusible radical intermediate in the case of APEX, or an activated biotin adenylate intermediate in the case of BioID and TurboID. After cell lysis, biotinylated proteins are harvested using streptavidin beads and identified by mass spectrometry.

PL has been applied in many cell types and species to map the proteome composition of organelles, including mitochondria (5, 8 ⇓ –10), synapses (11, 12), stress granules (13), and primary cilia (14). However, to increase the versatility of PL, new enzyme variants are needed. In particular, split enzymes could enable greater spatial specificity in the targeting of biotinylation activity, as well as PL activity that is conditional on a specific input, such as drug, calcium, or cell–cell contact. For example, contact sites between mitochondria and the endoplasmic reticulum (ER) mediate diverse biology, from lipid biosynthesis and Ca +2 signaling to regulation of mitochondrial fission (15). There is great interest in probing the proteomic composition of ER–mitochondria contacts. However, direct fusion of a PL enzyme to one of the known ER–mitochondria contact resident proteins (e.g., Drp1 or Mff) would generate PL activity outside of ER–mitochondria contacts as well, because these proteins also reside in other subcellular locations (16, 17). On the other hand, use of a split PL enzyme, with one fragment targeted to the mitochondria and the other targeted to the ER, would restrict biotinylation activity to ER–mitochondria contact sites specifically.

Split forms of APEX (18) and BioID (19 ⇓ –21) have previously been reported. However, split-APEX (developed by us) has not been used for proteomics, and the requirement for exogenous H2O2 and heme addition limits its utility in vivo. Split-BioID was first reported by De Munter et al. (19), followed by more active versions from Schopp et al. (20) and Kwak et al. (21). All are derived from the parental enzyme BioID, which requires 18 to 24 h of biotin labeling. We show below that the Schopp et al. (20) split-BioID does not produce detectable activity, while the Kwak et al. (21) split-BioID requires 16+ h of labeling to generate sufficient signal.

Hence we sought to develop an improved, more active split PL enzyme by starting from TurboID. In contrast to APEX, TurboID does not require any cofactors or cooxidants just biotin addition initiates labeling in cells or animals. TurboID is also >100-fold faster than BioID, requiring only 1 to 10 min of labeling time (7). We performed a screen of 14 different TurboID split sites to identify optimal fragments for high-affinity and low-affinity reconstitution. We converged upon TurboID split at L73/G74, which gave rapamycin-dependent reconstitution when fused to FRB and FKBP in multiple subcellular organelles. We then used this split-TurboID to perform proteomic mapping of ER–mitochondria contact sites in mammalian cells. The resulting proteome of 101 proteins is highly specific and identifies many new ER–mitochondria contact site candidates, eight of which we validated by biochemical fractionation or overexpression imaging.

In a human, what non-germline cells have the highest/lowest mass? - Biology

Figure 1: Back of the envelope calculation showing the fraction of the cell dry mass dedicated to ribosomes at a fast bacterial growth rate. Number of ribosomes based on BNID 101441 and cell dry mass based on BNID 103891.

Figure 2: Fraction of ribosomal protein synthesis rate out of the total cell protein synthesis. Measurements were performed on cultures in balanced growth and thus the relative rate is similar to the relative abundance of the ribosomal proteins in the proteome. Adapted from J. L. Ingraham et al., “Physiology of the bacterial cell” page 276, Sinauer 1990.

One of the familiar refrains in nearly all biology textbooks is that proteins are the workhorses of the cell. As a result, cells are deeply attentive to all the steps between the readout of the genetic information hidden within DNA and the expression of active proteins. One of the ways that the overall rhythm of protein production is controlled is through tuning the number of ribosomes. Ribosomes are one of the dominant constituents in cells and in rapidly dividing cells, they begin to take up a significant fraction of the cellular interior. The RNA making up these ribosomes accounts for ≈85% of the cell’s overall RNA pool (BNID 106421). Though DNA replication, transcription and translation are the three pillars of the central dogma, within the proteome, the fraction dedicated to DNA polymerase (BNID 104123) or RNA polymerase (BNID 101440) is many times smaller than the tens of percent of the cell protein dedicated to ribosomes (BNID 107349, 102345). As such there is special interest in the abundance of ribosomes and the dependence of this abundance on growth rate. The seminal work of Schaechter et al. established early on the far-from-trivial observation that the ribosomal fraction is a function of the growth rate and mostly independent of the substrate, that is, different media leading to similar growth rates tend to have similar ribosomal fractions (M. Schaechter et al. J Gen Microbiol. 19:592, 1958). Members of the so called “Copenhagen school” (including Schaechter, Maaloe, Marr, Neidhardt, Ingraham and others) continued to make extensive quantitative characterization of how the cell constituents vary with growth rate that serve as benchmarks decades after their publication and provide a compelling example of quantitative biology long before the advent of high throughput techniques.

Table 1 shows the number of ribosomes in E. coli at different doubling times. In the table it is also evident how the cell mass (and volume) depends strongly on growth rate, with faster dividing cells being much larger. As calculated in the fourth column of the table, and schematically in Figure 1, at a fast doubling time of 24 minutes the 72,000 ribosomes per cell represent over 1/3 of the dry mass of the cell (BNID 101441, 103891). Accurate measurements of this fraction from the 1970s are shown in Figure 2.

Table 1: Number and fraction of ribosomes as a function of the doubling time. Values are rounded to one significant digit. Ribosomes per cell are from “E. coli & Salmonella handbook”, Chapter 97, Table 3. Dry mass per cell is from E. coli & Salmonella, Chapter 97, Table 2. Ribosome dry mass fraction is calculated based on ribosome mass of 2.7MDa (BNID 100118).

Several models have been set forth to explain these observed trends for the number of ribosomes per cell. In order to divide, a cell has to replicate its protein content. If the translation rate is constant there is a neat deduction to be made. We thus make this assumption even though the translation rate varies from ≈20 aa/sec in E. coli at fast growth rate to closer to ≈10 aa/sec under slow growth (BNID 100059). Think of a given cell volume in the cytoplasm. Irrespective of the doubling time, the ribosomes in this volume have to produce the total mass of proteins in the volume within a cell cycle. If the cell cycle becomes say three times shorter then the necessary ribosome concentration must be three times higher to complete the task. This tacitly assumes that the polymerization rate is constant, that active protein degradation is negligible and that the overall protein content does not change with growth rate. This is the logic underpinning the prediction that the ribosomal fraction is proportional to the growth rate. Stated differently, as the doubling time becomes shorter, the required ribosomal fraction is predicted to increase such that the ribosomal fraction times the doubling time is a constant reflecting the total proteome concentration. The analysis also suggests that the synthesis rate scales as the growth rate squared, because the time to reach the required ribosome concentration becomes shorter in proportion with the doubling time. How well does this toy model fit the experimental observations?

Figure 3: Cryo-electron tomography of the tiny Spiroplasma melliferum. Using algorithms for pattern recognition and classification, components of the cell such as ribosomes were localized and counted. (A) Single cryo-electron microscopy image. (B) 3D reconstruction showing the ribosomes that were identified. Ribosomes labeled in green were identified with high fidelity while those labeled in yellow were identified with intermediate fidelity. (C) Close up view of part of the cell. Adapted from J. O. Ortiz et al., Journal of Structural Biology 156:334, 2006.

As shown in the right column of Table 1 and in Figure 2, the ratio of ribosome fraction to growth rate is relatively constant for the faster growth rates in the range of 24-40 minutes as predicted by the simple model above and the ratio is not constant at slow growth rates. Indeed at slower growth rates the ribosome rate is suggested to be slower (BNID 100059). More advanced models (e.g. M. Scott et al., Science, 330:1099, 2010) consider different constituents of the cells (for example, a protein fraction that is independent of growth rate, a fraction related to the ribosomes and a fraction related to the quality of the growth medium) that result in more nuanced predictions that fit the data over a larger range of conditions. Such models are a large step towards answering the basic question of what governs the maximal growth rates of cells.

Figure 4: Counting and localizing ribosomes inside cells using single molecule microscopy. (A) Two ribosomes identified from the full super-resolution image shown below. (B) Single-molecule intensity distribution. (C) Number of ribosomes as a function of cellular volume. (Adapted from S. Bakshi et al, Molecular Microbiology 85:21, 2012.)

Traditionally, measuring the number of ribosomes per cell was based on separating the ribosomes from the rest of the cell constituents, measuring what fraction of the total mass comes from these ribosomes and then with conversion factors based on estimations of cell size and mass, ribosomal molecular weight etc. inferring the abundance per cell. Recently a more direct approach is becoming available based on explicitly counting individual ribosomes. In cryo-electron microscopy, rapidly frozen cells are visualized from many angles to create what is known as a tomographic 3D map of the cell. The known structure of the ribosome is then used as a template that can be searched in the complete cell tomogram. This technique was applied to the small, spiral-shaped prokaryote Spiroplasma melliferum. As shown in Figure 3, in this tiny cell, 10-100 times smaller than E. coli by volume (BNID 108949, 108951) and slower in growth, researchers counted on average 1000 ribosomes per cell (BNID 108945). Similar direct counting efforts have been made using the super-resolution techniques that have impacted fluorescence microscopy as shown in Figure 4 where a count was made of the ribosomes in E. coli. A comparison of the results from these two methods is made in Figure 5 where a simple estimate of the ribosomal density is made from the cryo-electron microscopy images and this density is then scaled up to a full E. coli volume, demonstrating an encouraging consistency between the different methods.

Figure 5: Back of envelope estimate on how many ribosomes are in a cellular volume.

Science without gravity at the International Space Station

More than 3,000 such scientific tests have been carried out at the ISS since manned missions began in 2000

In two decades orbiting the Earth the International Space Station has become a cutting-edge cosmic laboratory, with astronauts researching everything from black holes to disease and even gardening in microgravity.

The ISS, which orbits about 250 miles above Earth, is as large as a football field inside and divided up like a beehive into spaces where the crew can carry out experiments with guidance from researchers on the ground.

Often, the astronauts are also the guinea pigs.

More than 3,000 scientific tests have been carried out at the ISS since its manned missions began in 2000.

"From a science perspective, there have been some major discoveries," said Robert Pearlman, space historian and co-author of "Space Stations: The Art, Science, and Reality of Working in Space".

The latest mission—named "Alpha" after Alpha Centauri, the closest star system to our own—will be no exception.

On Thursday, US astronauts Shane Kimbrough and Megan McArthur, the Japan Aerospace Exploration Agency's Akihiko Hoshide and the European Space Agency's Thomas Pesquet will blast off for the ISS aboard the SpaceX mission Crew-2.

They are likely to be busy.

Alongside work to maintain the space station itself, around a hundred experiments are in the diary for their six-month mission.

The new crew (from left to right): European Space Agency astronaut Thomas Pesquet, NASA's Megan McArthur and Shane Kimbrough, and Japan Aerospace Exploration Agency's Akihiko Hoshide

These include an acoustic technique using ultrasonic waves to move and manipulate objects or liquids without touching them.

France's Pesquet has said his favourite planned research is a study examining the effects of weightlessness on brain organoids—mini brains created using stem cell technology.

Scientists hope this research can eventually help space agencies prepare for distant space missions which will expose crews to the rigours of space for long periods of time, and even help fight brain disease on Earth.

"It really sounds like science fiction to me," joked Pesquet, an aerospace engineer.

There is ongoing research into what are known as "tissue chips"—small models of human organs that are made up of different types of cells and used to study things like ageing in the immune system, kidney function and muscle loss.

"We don't fully understand why, but in microgravity, cell-to-cell communication works differently than it does in a cell culture flask on Earth," said Liz Warren, senior program director at the ISS US National Laboratory, adding cells also gather together differently.

"These features allow cells to behave more like they do when inside the body. Thus, microgravity appears to provide a unique opportunity for tissue engineering."

Another important element of the mission is upgrading the station's solar power system by installing new compact panels that roll open like a huge yoga mat.

Crew-2's launch day coincides with Earth Day, and by the time the crew returns they will have also contributed to environmental research by taking 1.5 million images of phenomena like artificial lighting at night, algal blooms, and the breakup of Antarctic ice shelves.

The ISS offers new perspectives on Earth as well as Space

Experimental evolution

The experiments are designed for the long term, beyond individual missions, said Sebastien Barde of France's Cadmos, which organises microgravity science experiments in space.

The study of weightlessness—or microgravity—has gone from "pioneering to something standardised", with increasingly precise methods of measurement, Barde said.

"Twenty years ago, there was no ultrasound machine on board," he added.

Claudie Haignere, the first French woman to fly in space, visited the ISS in 2001 and remembers it as rather "poorly equipped".

Now she says she it boasts "exceptional laboratories".

The astronauts also stay longer—six months, versus a fortnight for the first manned flights—giving researchers more time to measure the effects of microgravity on them.

Spaceflight changes the human body.

Graphic on the International Space Station (ISS).

It weakens muscle and bone and affects the heart and blood vessels. Some of the effects resemble a speeded-up progression of ageing and diseases on Earth.

Whilst being guinea pigs for this research, the ISS crew has also collected data on black holes, pulsars and cosmic particles to help expand our understanding of the Universe.

With the ability to grow supplemental food seen as an important step to helping humans venture deeper into Space, they have even done some experimental gardening.

In 2015, astronauts sampled their first space-grown salad and they have since tried growing radishes.

Pearlman said the discoveries range from those related to human health—like a treatment for salmonella—to experimental engineering.

"One very promising technology right now that's just on the cusp of happening is 3D printing body parts," he said.

Some have raised concerns about the cost of the ISS, while NASA itself is seeking to disengage as its attention shifts to deeper space.

But Barde said the space station, scheduled to retire in 2028, is the only platform for some scientists to pursue their research, whether that is in medicine or material sciences that need an environment without gravity.

He dismissed the idea that we have learned everything we need to know: "It's like wondering if you really need to enlarge a telescope, because you have seen 'enough' stars!"

Perhaps you have a degree in biology, but aren’t sure if working in a science- or biology-related field is the right choice for you. In this case, you may need to sell your skills and experiences to employers who have little concern for the interplay of animal species or the interworking of the human body. Fortunately, you can still sell your useful skills to many employers.

During interviews, it’s important to focus on many of the specific projects you worked on, citing examples of problem-solving, leadership, group work, or any other skills that could apply to the career. Discuss your understanding of research, data analysis, and other skills that made you a successful biology student and you’ll be more attractive to more clients.

The Effect of Temperature on Rate of Reaction

As a rule of thumb, a rise in temperature of 10 °C doubles the reaction rate. Why might increasing the temperature alter the rate of a chemical reaction?

If you're thinking it's because the molecules involved are moving more quickly when the temperature is higher, you're on the right track. Temperature is actually a measure of the average kinetic energy of molecules in motion.

Molecules in motion tend to stay in motion until they encounter an external force, and when different reactant molecules are mixed together, they have little to run into besides each other.

When temperature increases, the amount of atomic or molecular collisions between molecules increases. But the change in reaction rate with temperature is not just a function of the temperature instead, temperature increases actually affect the rate constants (written k) of reactions in a predictable way.

5 White Blood Cells Types and Their Functions

White blood cells are also referred to as WBCs or leukocytes. They are the cells that make up the majority of the immune system, which is the part of the body that protects itself against foreign substances and various types of infections. Leukocytes are made in the bone marrow from multipotent cells called hematopoietic stem cells. Leukocytes exist in all parts of the body, including the connective tissue, lymph system, and the bloodstream. There are five different types of white blood cells, each of which has a different funtions in the immune system.

Five White Blood Cells Types and Their Functions

There are two different kinds of white blood cells and each looks different from one another under the microscope. These include granulocytes and agranulocytes.

  • Granulocytes have visible granules or grains inside the cells that have different cell functions. Types of granulocytes include basophils, neutrophils, and eosinophils.
  • Agranulocytes are free of visible grains under the microscope and include lymphocytes and monocytes.

Together, they coordinate with one another to fight off things like cancer, cellular damage, and infectious diseases. Below, detailed information about each type will be discussed.

1. Neutrophils

Neutrophils are the most common type of white blood cell in the body with levels of between 2000 to 7500 cells per mm 3 in the bloodstream. Neutrophils are medium-sized white blood cells with irregular nuclei and many granules that perform various functions within the cell.

Function: Neutrophils function by attaching to the walls of the blood vessels, blocking the passageway of germs that try to gain access to the blood through a cut or infectious area. Neutrophils are the first cells to reach an area where a breach in the body has been made. They kill germs by means of a process known as phagocytosis or &ldquocell-eating&rdquo. Besides eating bacteria one-by-one, they also release a burst of super oxides that have the ability to kill many bacteria at the same time.

2. Lymphocytes

Lymphocytes are small, round cells that have a large nucleus within a small amount of cytoplasm. They have an important function in the immune system, being major players in the humoral immune system, which is the part of the immune system that relates to antibody production. Lymphocytes tend to take up residence in lymphatic tissues, including the spleen, tonsils, and lymph nodes. There are about 1300 to 4000 lymphocytes per mm 3 of blood.

Function: B lymphocytes make antibodies, which is one of the final steps in disease resistance. When B lymphocytes make antibodies, they prime pathogens for destruction and then make memory cells ready that can go into action at any time, remembering a previous infection with a specific pathogen. T lymphocytes are another type of lymphocyte, differentiated in the thymus and important in cell-mediated immunity.

3. Monocytes

Monocytes are the largest of the types of white blood cells. There are only about 200-800 monocytes per mm 3 of blood. Monocytes are agranulocytes, meaning they have few granules in the cytoplasm when seen under the microscope. Monocytes turn into macrophages when they exit the bloodstream.

Function: As macrophages, monocytes do the job of phagocytosis (cell-eating) of any type of dead cell in the body, whether it is a somatic cell or a dead neutrophil. Because of their large size, they have the ability to digest large foreign particles in a wound unlike other kinds of white blood cells.

4. Eosinophils

There aren&rsquot that many eosinophils in the bloodstream&mdashonly about 40-400 cells per mm 3 of blood. They have large granules that help in cellular functions. Eosinophils are especially important when it comes to allergies and worm infestations.

Function: Eosinophils work by releasing toxins from their granules to kill pathogens. The main pathogens eosinophils act against are parasites and worms. High eosinophil counts are associated with allergic reactions.

5. Basophils

Basophils are the least frequent type of white blood cell, with only 0-100 cells per mm 3 of blood. Basophils have large granules that perform functions that are not well known. They are very colorful when stained and looked at under the microscope, making them easy to identify.

Function: Basophils have the ability to secrete anticoagulants and antibodies that have function against hypersensitivity reactions in the bloodstream. They act immediately as part of the immune system&rsquos action against foreign invaders. Basophils contain histamine, which dilates the vessels to bring more immune cells to the area of injury.

You can also learn types of white blood cells in greater detail from the video below:

Monitor Your White Blood Cell Counts

Your doctor will monitor your white blood cell count if there is evidence of infection or if you are on medication that may lower your white blood cell count. If you have an abnormal white blood cell count, you can have &ldquoleukopenia&rdquo, which means low white blood cell count, or &ldquoleukocytosis&rdquo, which is a high white blood cell count.

Leukopenia is a low white blood cell count that can be caused by damage to the bone marrow from things like medications, radiation, or chemotherapy. Folate or vitamin B12 deficiency can also result in it. So can lymphoma, in which cancer cells take over the bone marrow, preventing the release of the various types of white blood cells. HIV is another condition that can damage the production of white blood cells, leading to leukopenia.

Leukocytosis is a high white blood cell count that can be caused by a number of conditions, including various types of infections, inflammatory disease in your body, situations where are a high number of dead cells in the body, leukemia and allergies.

Concentrations of parabens in human breast tumours

Parabens are used as preservatives in many thousands of cosmetic, food and pharmaceutical products to which the human population is exposed. Although recent reports of the oestrogenic properties of parabens have challenged current concepts of their toxicity in these consumer products, the question remains as to whether any of the parabens can accumulate intact in the body from the long-term, low-dose levels to which humans are exposed. Initial studies reported here show that parabens can be extracted from human breast tissue and detected by thin-layer chromatography. More detailed studies enabled identification and measurement of mean concentrations of individual parabens in samples of 20 human breast tumours by high-pressure liquid chromatography followed by tandem mass spectrometry. The mean concentration of parabens in these 20 human breast tumours was found to be 20.6 ± 4.2 ng g −1 tissue. Comparison of individual parabens showed that methylparaben was present at the highest level (with a mean value of 12.8 ± 2.2 ng g −1 tissue) and represents 62% of the total paraben recovered in the extractions. These studies demonstrate that parabens can be found intact in the human breast and this should open the way technically for more detailed information to be obtained on body burdens of parabens and in particular whether body burdens are different in cancer from those in normal tissues. Copyright © 2004 John Wiley & Sons, Ltd.

Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (

-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, -Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse -Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions.

1. Introduction

Asthma is a chronic respiratory condition characterised by recurrent exacerbations [1]. A feature of asthma (especially severe asthma) is airway remodelling, that is, increased smooth muscle mass, fibrosis, and excessive mucus production [2]. The epithelium plays a key role in the development of airway remodelling and inflammation as it represents the primary barrier to environmental exposures and also signals to other cell types within the context of the epithelial mesenchymal trophic unit [3, 4].

In vitro models using primary cells and cell lines are essential for understanding the function of the epithelium relevant to asthma. Cells are routinely cultured in submerged monolayers on a plastic substrate. In order to obtain a more physiological model, primary human bronchial epithelial cells (HBECs) may be cultured at air-liquid interface (ALI) using defined medium to drive a differentiated phenotype [5]. This model shows a pseudostratified, polarised phenotype, including ciliated and goblet cells and develops high transepithelial electrical resistance (TEER) [6, 7]. Measurement of TEER provides an indirect measure of formation of tight junctions and is often used as a marker of disruption of the epithelial layer [8].

Cultured primary HBECs from asthma and non-asthma subjects have been compared in a number of studies, to investigate intrinsic differences in the asthmatic epithelium. Epithelial cells from asthmatic patients display differential expression of genes associated with inflammation, repair, and remodelling and have been shown to differ from normal cells in culture, including increased proliferation [9] and slower repair of a mechanical wound [10, 11]. Several groups have cultured asthmatic epithelial cells at ALI, showing a less differentiated phenotype, that is, increased numbers of basal cells [12] or decreased tight junction formation [13], and differing responses to stimulation including viral infection, mechanical wounding, and cigarette smoke [12–14]. There has been some debate regarding reported differences between normal and asthmatic cells. For instance, Hackett et al. [12] report no difference in TEER between normal and asthmatic cultures, whilst Xiao and colleagues suggest that cells from asthmatic subjects show decreased TEER and disrupted tight junctions [13]. These discrepancies may reflect differences in donor profile (donors were significantly older in the Xiao study), cell source (post mortem donor lungs versus bronchial brushings), or the much greater number of subjects included in the Xiao study. Paediatric asthmatic HBECs in monolayer culture show slower repair of a mechanical wound [10, 11]. At ALI, HBECs from asthma donors show increased cytokine release in response to mechanical wounding, or viral or particulate matter exposure [12], and are more sensitive to disruption of TEER by cigarette smoke extract [13]. Another study found that whilst HBECs from normal donors showed an increased rate of wound repair in response to IL-1β treatment, asthmatic cells did not show this response [14]. These results may suggest that asthmatic cells at ALI have an intrinsically different phenotype and show different signalling responses to normal cells and support the utility of epithelial cell culture in asthma research.

Direct comparisons of normal and asthmatic cells allow characterisation of the asthmatic phenotype however, they are less helpful when trying to dissect the underlying mechanisms behind epithelial changes in asthma. Normal primary bronchial epithelial cells and cell lines may be used to model various aspects of asthma. Cytokines may be added to cells in monolayer or ALI culture [15–17], whilst asthma triggers such as Derp1 or rhinovirus have been applied to the cells to mimic allergen inhalation or viral exacerbation [18, 19]. Danahay et al. treated ALI HBECs with IL-13 or IL-4, resulting in changes in permeability, suggesting that these asthma-related cytokines may contribute to a more secretory phenotype [15], whilst Wadsworth and colleagues found that addition of IL-13 and other

cytokines led to increased MMP7 and FasL release, which may lead to epithelial damage and inflammation [16]. In another study, HBEC or BEAS-2B cells at ALI were treated with leukotriene D4, resulting in signalling via EGFR and release of IL-8 [17]. Overall these data demonstrate that the use of HBEC and cell line cultures can provide a unique insight into mechanisms underlying asthma and it is important to understand the strengths and weaknesses of these culture systems.

Accumulating data suggest that bronchial epithelial cells may be a viable drug target in asthma [20]. Cell culture models are used in drug development, both to assess the direct effect of potential drugs on cell function and signalling and to investigate drug uptake and metabolism [21]. Although primary cells are the gold standard, there are some disadvantages to their use including cost, limited life span, and variability between donors, passage, or experiments. Primary cells may also be more difficult to transfect or otherwise manipulate. This has led to the use of cell line systems, in both monolayer culture and at ALI. The Calu-3 cell line was established from a pleural effusion of a lung adenocarcinoma, derived from submucosal gland serous cells [22–24]. It is often used at ALI as a model system, particularly for investigations of tight junction and barrier formation [23], for instance, showing that rhinovirus infection leads to decreased TEER and increased permeability [19]. The BEAS-2B cell line, originally developed by immortalization of normal human bronchial epithelial cells using AD12-SV40 virus [25], has been less frequently used at ALI however there is some literature using BEAS-2B in this system [17, 26]. Although these cells have been separately characterised by techniques such as immunofluorescence and TEER, no systematic comparison of primary cell and cell line culture models in this system has been reported.

The aim of the current study was to evaluate primary and non-primary bronchial epithelial cell culture systems as (ALI-) differentiated models for asthma research. We cultured primary HBECs from two donors, Calu-3, BEAS-2B, and BEAS-2B R1 (a subclone of BEAS-2B, cultured in the presence of foetal calf serum (FCS)) in their respective media at ALI. Development of TEER was measured over 28 days, RNA was collected at days 7, 14, and 21, and immunofluorescence was performed at day 28. We measured expression of a panel of differentiation (β-Tubulin IV, a ciliated cell marker, and MUC5AC, a goblet cell marker [27]) and tight junction/adhesion (E-Cadherin and ZO-1) markers by real-time quantitative PCR (qPCR) and confocal imaging to allow direct comparison of the phenotype of these different cell systems.

We show that although primary cells develop a differentiated phenotype, their TEER is highly variable, confirming the need to use multiple experiments and donors in primary cell systems. Calu-3 cells showed high TEER and similar expression of markers compared to primary cells, suggesting that these cells may be the most suitable model cell line for ALI experiments. Our work (1) indicates that all model systems, including primary cells, should be validated to ensure that the most suitable model is being used for a specific research question and (2) highlights the difficulties in utilising primary cells in epithelial cell research.

2. Materials and Methods

2.1. Cell Culture and ALI Differentiation

Human bronchial epithelial cells (NHBEC, Lonza, Wokingham, UK) were expanded in growth factor-supplemented medium (BEGM, Lonza) and differentiated at (ALI) at passage 3-4 in differentiation medium (BEDM) according to a previously published method [15, 28]. BEDM was composed of 50 : 50 Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) : BEBM (Lonza) with Lonza singlequots, excluding triiodo-L-thyronine and retinoic acid, but including GA-1000 (Gentamicin and Amphotericin-B). BEDM was supplemented with 50 nM retinoic acid at time of use. All experiments were performed using a single lot of BEBM and singlequots to avoid batch variation. Medium was used within one month of preparation, as recommended by Lonza.

Calu-3 lung adenocarcinoma cells [22] (obtained from ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) (Sigma) supplemented with 10% FCS, 1% MEM non-essential amino acid solution (Sigma), and 1% Penicillin/Streptomycin (Sigma). BEAS-2B [25], a transformed bronchial epithelial cell line (gift from Dr R. Clothier, University of Nottingham), was cultured in BEGM and differentiated at ALI in BEDM. BEAS-2B R1 [29] (a subclone of BEAS-2B) (gift from Dr. R. Penn, University of Maryland, Philadelphia, PA) was cultured in DMEM supplemented with 10% FCS and 1% Penicillin/Streptomycin. All cells were cultured on 12 mm polyester Transwell inserts with a pore size of 0.4 μm (Corning NY, USA). Cells were plated at 100,000 cells per insert in appropriate medium. When confluent (

3 days), cells were raised to ALI. Medium was replaced and the apical face washed with phosphate-buffered saline (PBS) every 48 hours. RNA was extracted after 7, 14, and 21 days at ALI and cells were fixed for immunostaining after 28 days at ALI.

2.2. Transepithelial Electrical Resistance (TEER)

The transepithelial electrical resistance (TEER) was measured in differentiating cells using an EVOM2 epithelial volt-ohm meter (World precision Instruments UK, Stevenage), over 21 to 28 days at ALI to confirm development of tight junctions. Briefly, medium was aspirated and replaced with 1 mL in the basolateral and 0.5 mL in the apical compartment. Cultures were equilibrated in the incubator for 30 minutes before measurement of TEER. Apical medium was then aspirated to restore ALI. TEER of insert and medium alone was subtracted from measured TEER and Ω·cm 2 calculated by multiplying by the insert area.

2.3. Immunofluorescence of Cultured Cells

ALI cultured cells were fixed in situ on inserts and transferred to glass slides for visualisation. Cells were fixed using 4% formaldehyde and blocked/permeabilised with PBS, 10% goat serum, 1% BSA, and 0.15% Triton-X. Cells were incubated with appropriate primary antibodies at 4°C overnight (Table 1), and FITC or rhodamine-TRITC labelled secondary for 1 hour at room temperature before mounting in HardSet DAPI (Vector Labs). Controls were incubated with secondary antibody alone or primary isotype control antibody followed by secondary antibody. Cells were visualized using the Zeiss spinning disk confocal microscope using Volocity software (version 5.5, PerkinElmer, Cambridge, UK).

2.4. Quantitative PCR (qPCR)

Cultured cells were lysed and RNA was extracted using silica columns (RNeasy mini kit, Qiagen, Crawley, UK). cDNA was synthesized using Superscript II (Invitrogen, Paisley, UK) and random hexamer primers as per instructions. mRNA levels were quantified using a series of TaqMan assays (Table 2). Probes were labelled with FAM and TAMRA. qPCR was performed using TaqMan gene expression master mix (Applied Biosystems, Warrington, UK) and HPRT1 (4310890E, Applied Biosystems) endogenous control on a Stratagene MxPro3005 machine using 40 cycles of 95°C 15 sec, 60°C 60 sec. Data were normalised using the housekeeper (HPRT1) and the

3. Results

3.1. Primary Epithelial Cells and Cell Lines Develop TEER When Cultured at ALI

Primary HBECs (2 donors) and cell lines were cultured at ALI and TEER measured every 2-3 days for 21–28 days (Figure 1, Table 3). All primary cell experiments were performed at passage 3-4 from different frozen vials. Experiments performed at passage three (two in Donor 1, one in Donor 2) developed TEER >350 Ω·cm 2 , whilst experiments performed at passage four developed TEER <150 Ω·cm 2 (Figures 1(a) and 1(b)). Calu-3 (passage 35–37) developed maximum TEER >400 Ω·cm 2 in all experiments, reaching a peak between days 9 and 12. Thereafter, values dropped slightly before reaching a more variable plateau (Figure 1(c)). BEAS-2B reached a maximum TEER of 100–150 Ω·cm 2 by around day 14 (Figure 1(d)), regardless of passage, whilst BEAS-2B R1 did not develop significant TEER (Figure 1(e)).

(a) HBEC D1
(b) HBEC D2
(c) Calu-3
(d) BEAS-2B
(e) BEAS-2B R1
(a) HBEC D1
(b) HBEC D2
(c) Calu-3
(d) BEAS-2B
(e) BEAS-2B R1 Development of transepithelial electrical resistance (TEER) in cells grown at (ALI). Different primary cells and cell lines were cultured at ALI over 21–28 days. TEER was measured every 2-3 days. Results from three separate experiments are shown for each cell line/donor, six replicates per experiment. HBEC D1 is Donor 1 and HBEC D2 is Donor 2. Error bars show standard deviation.
3.2. Primary Epithelial Cells and Cell Lines Show Morphological Differences

The different cells used in this study showed different phenotypes in culture. Phase contrast images give a limited indication of these differences however gross morphological differences are present (Figure 2). Calu-3 cells took longest to become fully confluent, probably due to their tendency to form discrete colonies, unlike the other cells which form a more even monolayer. HBECs showed darker areas of denser (probably more stratified) cells and lighter, less dense areas (Figures 2(a) and 2(b)). The Calu-3 cell phenotype was more homogenous (Figure 2(c)), with increased mucus secretion apparent on washing. BEAS-2B cells consistently developed an apical layer of material which was not removed by washing (Figure 2(d)). BEAS-2B R1 cells had a very homogenous appearance, with no indication of mucus production or differentiation (Figure 2(e)).

(a) HBEC D1
(b) HBEC D2
(c) Calu-3
(d) BEAS-2B
(e) BEAS-2B R1
(a) HBEC D1
(b) HBEC D2
(c) Calu-3
(d) BEAS-2B
(e) BEAS-2B R1 Phase contrast images of cells at ALI. Different primary cells and cell lines were cultured at ALI over 21–28 days. Phase contrast images were taken at 21 days. Representative images are from three independent experiments.
3.3. Primary Epithelial Cells and Cell Lines Express Characteristic Differentiation Markers

At 28 days, cells were fixed and immunostained with antibodies specific for β-Tubulin IV and MUC5AC (Figure 3, Table 3). Although β-Tubulin IV is often expressed as a cytoskeletal protein, apical expression is a commonly used marker of ciliated epithelial cells [27]. MUC5AC is expressed by goblet cells as a component of mucus. Single image slices and z-stacks are shown to give an indication of the overall level of expression and location in the cell layer (basal versus apical). As ALI culture thickness varied between cell types, the brightest image is shown in each case. These were representative of 2-3 experiments per donor or cell type. HBEC images shown for both donors are from experiments reaching low TEER however, β-Tubulin IV and MUC5AC expression did not seem to reflect TEER values (data not shown). HBECs presented apical β-Tubulin IV expression in a subset of cells with a greater proportion of cells from Donor 1 than Donor 2 showing expression. β-Tubulin IV expression was observed in Calu-3 layers but staining was only apparent below the apical pole of the cells. Strong staining for β-Tubulin IV was obtained at the apical side of BEAS-2B cells. BEAS-2B R1 showed apical staining in a subset of cells. While both HBEC donors and Calu-3 cells was stained positive for MUC5AC expression in a subset of cells towards the apical side of the cell layer, neither BEAS-2B subtypes showed significant MUC5AC staining.

Immunofluorescent confocal imaging of differentiation markers. Localisation patterns of β-Tubulin IV, MUC5AC, and the Mouse IgG Isotype control at 28 days ALI were evaluated as described in the methods section. Single Z-slices are shown representing maximum intensity observed, with the corresponding Z-stack image below for β-Tubulin IV and MUC5AC. Scale bar represents 50 μm. Representative images are from three independent experiments. HBEC images were taken from experiments with low TEER (Figure 1).
3.4. Primary Epithelial Cells and Cell Lines Express Tight Junction Proteins

Sections were costained for expression of ZO-1 (a tight junction protein) and E-Cadherin (a cell adhesion molecule and epithelial cell marker). Matched, single confocal slices and z-stacks are shown. The brightest image from each stack was chosen to allow comparison of maximum expression in each cell culture system (Figure 4, Table 3). Images are representative of 2-3 experiments per donor or cell type. Both HBEC donors showed strong staining for ZO-1 that was localised to cell membranes/cell-cell junctions. This staining may be stronger in Donor 1 (where TEER reached >350 Ω·cm 2 ) than Donor 2 (where low TEER <150 Ω·cm 2 was reached) however the difference in staining was slight, compared to the variation in TEER. Overall, ZO-1 staining was performed in five HBEC experiments and no correlation between staining and final TEER was observed (data not shown). ZO-1 expression was weaker in Calu-3 cells, despite their consistently high (>300 Ω·cm 2 ) TEER, although similarly localised around cell boundaries. In both BEAS-2B subtypes, ZO-1 expression was generally diffuse however, BEAS-2B cells showed membrane localised expression in the apical cell layer. Both BEAS-2B subtypes also showed high non-specific staining with the rabbit isotype control, suggesting that ZO-1 protein levels may be lower than they appeared. E-Cadherin expression was seen in all cells except BEAS-2B R1. In both HBEC donors and Calu-3, expression was tightly localised to the cell membrane/cell-cell junctions, whilst in BEAS-2B expression was more diffuse in the basal layer, but membrane was localised in the apical layer.

Immunofluorescent confocal imaging of tight junction proteins. Localisation patterns of ZO-1, E-Cadherin, and the Rabbit IgG Isotype control at 28 days ALI were evaluated as described in the methods section. Images shown are single Z-stack slices representing maximum intensity observed with the corresponding Z-stack image below and are of matched fields using dual staining. The corresponding Mouse Isotype control for E-Cadherin can be seen in Figure 3. Scale bar represents 50 μm. Representative images are from three independent experiments. Images for HBEC Donor 1 were taken from an experiment reaching high TEER (>350 Ω·cm 2 ), whilst Donor 2 images were taken from an experiment reaching low TEER (Figure 1).
3.5. Expression of Differentiation and Tight Junction Markers Varies at the mRNA Level

Cells were harvested for RNA at days 7, 14, and 21 during ALI differentiation and qPCR performed for MUC5AC, β-Tubulin IV, E-Cadherin, and ZO-1 (Figure 5, Table 3). Representative data from one of two experiments are shown. HBEC results were taken from experiments in which TEER reached >350 Ω·cm 2 (Donor 1) and low TEER (Donor 2), whilst in replicate experiments, TEER >350 Ω·cm 2 was reached in both donors. Expression levels of all genes were significantly different between cell types, although not between HBEC donors (

for all genes, 2-way ANOVA). These effects were conserved in a second independent experiment. Overall, no replicated trends in gene expression over time were observed.

(a) MUC5AC
(b) β-Tubulin IV
(c) E-Cadherin
(d) ZO-1
(a) MUC5AC
(b) β-Tubulin IV
(c) E-Cadherin
(d) ZO-1 mRNA expression of differentiation and tight junction markers. Primary cells and cell lines were cultured at ALI over 21–28 days. RNA was extracted at days 7, 14, and 21 during ALI differentiation for each cell line or donor. Expression of MUC5AC (a), β-Tubulin IV (b), E-Cadherin (c), and ZO-1 (d) was measured. Data are normalised to the housekeeping gene HPRT1. Data are representative of two independent experiments. Error bars show standard deviation. Yellow, red, and blue bars represent expression at days 7, 14, and 21 post-ALI, respectively.

MUC5AC mRNA was similar in HBEC and Calu-3 cells. Although expression of MUC5AC appears to increase at later time points in the experiment shown ( , ANOVA), this effect was not conserved in a second independent experiment. MUC5AC mRNA was not detected in the two BEAS-2B subtypes (Figure 5(a)), consistent with immunofluorescence results. β-Tubulin IV expression was highest in BEAS-2B R1>Calu-3>BEAS-2B>HBEC (Figure 5(b)). This is in contrast to the immunofluorescence data where staining was lowest in BEAS-2B R1. Expression of E-Cadherin was highest in HBEC>Calu-3 and BEAS-2B>BEAS-2B R1 (not detected) (Figure 5(c)), whereas immunofluorescence was similar in HBEC and Calu-3. ZO-1 expression (Figure 5(d)) was highest in HBEC>BEAS-2B and BEAS-2B R1>Calu-3.

4. Discussion

We have evaluated two primary human donors of bronchial epithelial cells and Calu-3, BEAS-2B, and BEAS-2B R1 cell culture systems as ALI models of the airway epithelium for asthma research. For the first time, cell lines were directly compared to primary cells (Table 3). Using measurement of TEER [8], immunofluorescent staining, and qPCR, we have investigated formation of tight junctions (ZO-1 and E-Cadherin) as well as expression and localisation of suggested markers of ciliated (β-Tubulin IV) and goblet (MUC5AC) cells [27]. The main outcomes of our study are that (1) primary HBECs demonstrate a variable differentiated phenotype with the development of tight junctions and TEER showing experiment, passage, and donor variation, (2) Calu-3 cells exhibit many of the features of primary cells but have distinct differences including, for example, ZO-1 expression, and β-Tubulin IV localisation, although data generated were more reproducible, and (3) as anticipated, the BEAS-2B cell lines have limited differentiation capacity in ALI models. These data have implications for the use of both primary cells and cell lines for airway epithelial research in asthma.

The use of primary HBECs in vitro has provided insight into the potential mechanisms underlying asthma. This is exemplified by the recent findings of Xiao and colleagues [13], demonstrating that asthmatic epithelial cells at ALI show disrupted tight junctions and increased macromolecular permeability, reflecting the ex vivo phenotype. Another study by Hackett et al. observed an increased cytokine response to particulate matter, viral exposure, or mechanical wounding [12], demonstrating that asthmatic cells may show an aberrant inflammatory response to common environmental stimuli. Primary and cell line systems also play a role in dissecting the signalling networks involved in asthma. Normal HBECs at ALI treated with cytokines, for instance, show a potentially more secretory phenotype [15], whilst in HBEC or BEAS-2B cells, leukotriene D4 signals via EGFR to release IL-8 [17]. It is beyond doubt that these epithelial ALI culture systems show utility in asthma research therefore in the current study, we aimed to provide a direct comparison of a number of cell culture systems used in asthma research to help in selection of appropriate systems for specific research questions.

We measured expression of various proteins at the mRNA and protein levels as markers of differentiation. Although β-Tubulin IV is widely expressed in cultured cells, apical expression is often used to identify ciliated epithelial cells at ALI [27, 30], whilst MUC5AC is a mucus protein, expressed by goblet cells in the lung epithelium [31]. ZO-1 and E-Cadherin were included as markers of tight junction formation and barrier integrity. An alternative method of characterising ALI cultures is sectioning and performing histochemical analysis to confirm differentiation which gives a clearer indication of the multilayer structure (e.g. [12]). This study is limited by the use of immunofluorescence only however we can obtain an overview of the phenotypes of different systems using this method.

In this study, we found that mRNA expression was not tightly linked to immunostaining, particularly for β-Tubulin IV, where HBECs showed very low mRNA levels but high protein expression. This suggests that mRNA expression may not be a good marker of functional status for these genes. Differences between mRNA and protein levels may reflect experimental or biological issues [32, 33]. In our study, samples were taken for qPCR at days 7–21 and for immunofluorescence at day 28, a limitation which may partially explain these differences. Some variation in immunofluorescence between samples may reflect different protein localisation that is, diffuse faint staining may reflect similar amounts of protein to bright, localised staining. At the biological level, differences between mRNA and protein may reflect variation in posttranscriptional mechanisms between the cell types, such as mRNA stability or protein synthesis and turn-over.

When culturing primary cells at ALI, the choice of medium is very important, with different media delivering different degrees of stratification and cell phenotypes [5, 34]. We use “Gray’s medium” (BEDM), which is reported to allow development of a pseudostratified, polarised phenotype, including ciliated and goblet cells. This was confirmed in our hands, with localised expression of E-Cadherin, MUC5AC, and β-Tubulin IV observed in both primary cell donors. This model is reported to develop TEER [6, 7]. We found that development of TEER was variable. TEER >350 Ω·cm 2 was obtained in the three experiments performed at passage three, whilst TEER <150 Ω·cm 2 was obtained at passage four, despite consistent expression of ZO-1 mRNA and protein, localised to the cell membrane/cell-cell junctions. These observations reinforce the assumption that localised ZO-1 staining is not a surrogate marker for TEER and vice versa, as well as the importance of passage when using primary cells. The variation seen in this study between different experiments in a single donor is indicative of the potential issues when comparing normal versus asthma cells. Routinely, a single experiment is performed per donor [12, 13]. It is important that these experiments are performed with cells cultured for the same period of time and in the same batch of medium to minimise experimental variation.

The Calu-3 cell-line was established from a pleural effusion of a lung adenocarcinoma, derived from submucosal gland serous cells [22–24]. Calu-3 cells are routinely cultured in FCS-supplemented media [23] and spontaneously differentiate at ALI to give significant TEER [23, 35]. These cells are reported to express ZO-1 (a tight junction protein) and E-Cadherin (an epithelial marker and cell adhesion protein). We showed development of TEER >400 Ω·cm 2 and some expression of ZO-1 and E-Cadherin. Interestingly, although TEER was higher and more robust than in the primary HBECs, ZO-1 expression at both the mRNA and protein levels was lower, demonstrating that other tight junction proteins have a role to play in maintaining TEER. The cells expressed apical MUC5AC, as anticipated from their known secretory phenotype. However, staining for β-Tubulin IV expression was diffuse and not clearly located at the apical side, suggesting that villi or cilia had not formed in the Calu-3 model, in accordance with previous studies [23] that have shown that ciliated cells are sparse in the Calu-3 cell line.

The BEAS-2B cell line was originally developed by immortalization of normal human bronchial epithelial cells using AD12-SV40 virus [25]. This parental population of cells (as well as subclone S6, not used here) retains the ability to undergo squamous differentiation in response to TGFβ1 or serum [29]. The BEAS-2B R1 line was derived from the parental population by subculture in the presence of 5% FCS. Unlike the parental cell line, these cells are induced to proliferate by serum or TGFβ1 and have a more fusiform appearance [29]. BEAS-2B cells (S6 subclone, similar to the parental population) have previously been shown to attain TEER >100 Ω·cm 2 at higher passage, in KGM (keratinocyte growth medium, Clonetics) when supplemented with calcium [26], or when grown in BEGM [36] or Laboratory of Human Carcinogenesis (LHC) serum-free medium [37]. We used BEDM to drive BEAS-2B towards a differentiated phenotype. These cells attained the reported TEER of >100 Ω·cm 2 . At ALI, these cells developed an apical layer which stained strongly for β-Tubulin IV and showed localised ZO-1 and E-Cadherin staining. However, staining was fainter and more diffuse in the basal layer. It may be the presence of this apical layer that increases the TEER, rather than tight junction formation throughout the culture model. These cells did not express MUC5AC.

There is no literature regarding the use of the BEAS-2B R1 cell line at ALI therefore these cells were included essentially as a negative control, cultured in DMEM with 10% FCS. As anticipated, these cells did not develop TEER and expressed minimal levels of E-Cadherin at the RNA and protein level. The cells show apical β-Tubulin IV expression, but no MUC5AC or localised ZO-1 expression. The reduced E-Cadherin expression of this cell line suggests that they may have developed a more mesenchymal phenotype by culturing in the presence of FCS.

5. Conclusions

Normal and asthmatic primary bronchial epithelial cells and cell lines are widely used in asthma research. ALI models are used to attempt to more closely replicate the in vivo situation. We have evaluated primary bronchial epithelial cells from two donors and three cell lines in an ALI model with respect to various markers of differentiation. Although primary cells are regarded as the most physiologically relevant, they exhibit a high degree of variability between donors, experiments, and passage, particularly with respect to development of TEER. Primary cells are costly and therefore unsuitable for large scale experiments such as drug screening they also have a finite lifespan and may be difficult to manipulate. Cell lines may, therefore, present an attractive alternative model. We found that Calu-3 cells develop a high TEER and have a pattern of expression of epithelial markers similar to primary cells. Although frequently used in monolayer culture, the two BEAS-2B cell lines did not perform well in the ALI model, showing poor TEER and lacking expression of epithelial differentiation markers.

This work underlines the importance of using a well-characterised model, suitably validated for the outcomes of interest in any experiment. Importantly, this study highlights some of the challenges ahead characterising primary human airway epithelial cells from asthma and control donors accounting for the inter- and intradonor variability identified in the current study.


This work was funded by Asthma UK Grants 08/017 and 10/006 to Ian Sayers. C. E. Stewart and E. E. Torr equally contributed to the paper.


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