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Chunks of cells after trypsinization

Chunks of cells after trypsinization



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Recently I've came across a problem after trypsinization of my stably transfected CHO-cells. When I add medium to rinse off the cells from the bottom of the flask, there are usually big chunks of cells, which I almost cannot resuspend properly… I also tried to increase the incubation time with trypsin, but it did not help much.

Does anyone have a solution for this? The trypsin I use is the same batch as usual and in the meantime I also thawed 2 different freezing stocks, but I still have the same problem.

I also tried different pipettes, from 25 to 5mL. But I can pipette like a maniac up and down and up and down… still there are big chunks of cells floating around…


Gibco has an anti-clumping agent verified for CHO cells, as does Lonza, but I like Gibco reagents more. Cells tend to clump for a number of reasons, including extracellular DNA from lysed cells and the presence of Ca and Mg ions (important ions for cell adhesion and aggregation).

You could try these:

1) Wash and resuspend in balanced salt solution without magnesium or calcium. Like Hank's balanced salt solution.

or

2) Treat with DNase I. I think you could go as low as 1 IU/mL for 30min-1hr to bust up a fair amount of DNA clumping. You can do this in a basal medium (like RPMI, DMEM, etc.), or the balanced salt solution. Your medium can contain serum only if the serum has been heat-inactivated as proteases in the serum with inactivate your DNase. Incubate with DNase under gentle agitation at 37°C (I always had a tube rotator in an incubator for this purpose, just put your cells in a 50mL conical and set them to agitate for 1hr. You could also use a heated shaker unit). If you don't have this setup, you can agitate at room temp, but for a little longer perhaps.

and maybe

3) Once of the other obvious choices is to avoid passaging your clumping cells and collect the rest. You can do this by putting all your cells into a flask incl. the clumps, agitate for a moment, and let it sit so that the chunks quickly fall to the bottom, and collect everything but the stuff that fell to the bottom.


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Lab grown meat from tissue culture of animal cells is sustainable, using cells without killing livestock, with lower land use and water footprint. Japanese scientists succeeded in culturing chunks of meat, using electrical stimulation to cause muscle cell contraction to mimic the texture of steak.

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I wonder - does lab grown meat remove the ethical argument for vegetarians?

I wanna listen to that debate.

As a vegan, I think the answer has to be “Yes - the ethical issue has been removed”.

I appreciate that a biopsy of some kind is initially required, but I presume that once the first tissue sample has been taken it can continue to be used near indefinitely.

So whilst I guess there is still animal harm at the start, this is really negligible in the fullness of time especially when balanced with the benefits it brings.

Vegans are not, or at least should not be, unaware that vast numbers of insects, mice, etc. are killed in ordinary crop production which is unavoidable and accepted. Most rational animal ethics arguments are not about the complete abolition of animal suffering but the avoidance where possible.

It depends on what you mean by "the" ethical argument, because there are multiple ethical reasons for reducing or eliminating meat consumption.

This is probably wondered a lot more by non-vegetarians than vegetarians.

I've heard one vegan say no.

I've heard another say "Yes, as long as it doesn't have the same environmental impact"

Some might argue that it's still harmful to animals due to the conditions they are raised or if a live biopsy is used to obtain the animal cells, but presumably they could collect animal cells without hurting the animals and without killing them, so I am sure that a lot of vegetarians would be okay with this. Simply not all of them.

I think it's important to understand that vegetarians aren't all one people. I would go mad for this stuff, but I know people that are veggie who wouldn't.

From ones I've talked to, they have no issue with it. Only thing they bring up is how the initial cells are obtained, which is fair enough.

Yes. Although for me, I eat veggies because I think they taste better

I'm somewhat of a vegetarian, but not for the usual reasons. I'm Jewish and keep kosher. Kosher meat is so insanely expensive that it's actually cheaper to be vegetarian than to buy kosher meat. (Especially during the pandemic when I'm limiting how many stores I shop in since not every store carries kosher meat.)

Iɽ be curious as to whether this meat is deemed kosher. Usually, there are strict rules that need to be followed - from how the animal is raised, to its health, to how it's slaughtered. Most of these would moot points when it came to lab grown meat. Assuming the original cow was raised properly and in good health, would the entire line of meat be good? Obviously, there wouldn't be any illnesses to worry about. (Contaminants would be kept out of the growing environment.) Also, slaughter wouldn't be an issue since it's not really "alive" in the same way that a cow is. It would be interesting to see whether this makes really inexpensive kosher meat.


Chunks of cells after trypsinization - Biology

Isolation of Mouse Lung Endothelial Cells (MLEC)/ Mouse Heart Endothelial Cells (MHEC)

  • Fibronectin-coated T-75 flasks prepared by wetting for >1 hr. with 2 mL of human FN (Gibco #33016-015, 2.5ug/mL, sterile filtered, PBS, $.20/flask). One T-75 for two mice and for each passage.
  • 0.1% Gelatin coated flasks can also be used for plating the cells. (Do not use
    gelatin if the cells are used for column chromatography experiments. We have found that the gelatin clogs the columns).
  • Isolation Medium = Hi Glucose DMEM + 20%FCS + Pen/Strep.
  • Growth Medium = same + 100ug/mL Heparin (Sigma H-3933) + 100 ug/mL
    ECGS (Biomedical Technologies, Stoughton, MA, Cat#BT-203) +1x non-essential amino acids + 2mM L-glutamine + 1x sodium pyruvate + 25mM HEPES.
  • Instruments for animal dissection (sterilized in 70% EtOH): two scissors, two forceps.
  • Instruments for tissue dissociation (autoclaved): small forceps, small scissors, and 6&rdquo-long 14 gauge metal cannula (Fisher #1482516N), and B-D Falcon cell strainer 70um pore size (Fisher #08-771-2).
  • Type I Collagenase (Worthington) from Core, dissolved 2mg/mL (150-170 U/mg) in 25 mL of DPBS + Ca/Mg &rarr warm at 37 C, 1 hr. &rarr 0.22 um Sterile filter.
  • Dynabeads M-450 Sheep anti-Rat IgG (Dynal #110.07) in PBS + 0.1% BSA + 0.02% NaN3. Concentration: 4 x 10 8 beads/mL.
  • Purified Rat anti-Mouse CD102 (ICAM-2) antibody (Pharmingen #553325), 1mg / 1mL.
  • Purified Rat anti-Mouse CD31 (PECAM-1, clone MEC13.3) antibody (Pharmingen #553369), 0.5mg / 1 mL.
  • DPBS- without Ca 2+ and Mg 2+

    Preparation of Anti-mouse ICAM-2 (PECAM-1) Dynabeads:

  1. Resuspend and aliquot of beads in 2 mL of PBS + 0.1% BSA. Mix well.
  2. Place tube on magnetic separator (e.g., Dynal MPC-S) and leave for 1-2 min.
  3. Remove supernatant and repeat wash 3x.
  4. Resuspend beads to original volume with PBS + 0.1% BSA.
  5. Add 5 uL of purified Ab for each 100 uL of beads.
  6. Incubate overnight on rotator at 4°C (or 2hr at RT).
  7. Repeat wash as in Steps 1 &ndash 3 (4x).
  8. Resuspend beads in original volume with PBS + 0.1% BSA to maintain beads at 4x10 8 beads/mL.
  9. Store beads at 4°C and use within 1-2 weeks.
  10. 0.02% sodium azide may be added as preservative, but remember to wash thoroughly before use.

  1. Sacrifice a young adult mouse by CO2 inhalation.
  2. Soak skin with 70% EtOH.
  3. Nick skin above the pubis with scissors and deglove the skin to above the chest.
  4. Pin mouse supine on a Styrofoam board on a bench top. Remove any loose fur from gloves or field.
  5. Cut open anterior abdomen, and take down the anterior diaphragm with sterile scissors.
  6. Grasp lobes of the lung and heart and cut free from the mediastinum (with a new set of scissors and tweezers).
  7. Place lobes/heart in 15 mL cold Isolation Medium in a 10cm dish.
  8. Repeat Steps 1 &ndash 7 for a second mouse. Combine lobes from two mice.

  1. Transfer the dish with lung lobes to a sterile laminar flow hood for subsequent work.
  2. With new sterile scissors, cut off the heart (taking care to avoid as much of the aorta as possible). Cut the heart in half and place in 50 mL tube containing 40 mL of cold isolation media. Gently agitate.
  3. Then carefully cut out the lung lobes from any visible bronchi and mediastinal connective tissue.
  4. Put lung lobes in a dry 10cm dish and mince finely with scissors for 1 min.
    (Repeat for heart in a separate dish).
  5. Put lung pieces in 25 mL of pre-warmed, 37°C Collagenase (2 mg/mL). Wash all bits off the plate. (Repeat same for heart).
  6. Incubate at 37°C with gentle agitation for 45 min. (shaking waterbath or inversion in a hybrid oven).
  7. Using a 30 cc syringe attached firmly to the cannula, triturate the suspension 12 times, taking care to avoid frothing.
  8. Let chunks settle. Pipette suspension through a 70um disposable cell strainer (Falcon #35 2350) into a 50mL conical tube. Wash sieve with 10 mL of Medium.
  9. Spin down cell suspension at 400g (1300 rpm in GH3.7 rotor), 8 min., 4°C.
  10. Resuspend in 2 mL of cold PBS + 0.1% BSA, Leave behind any stubborn clumps.
  11. Count nucleated cells (e.g., count on a Coulter counter by adding 5 uL of suspension to 10mL of Isotonic buffer + 3 drops of Zap-globin).

  1. Resuspend the heart cells in 1 ml DPBS- and lung cells in 2 ml of DPBS-. (Lung &ndash 1ml/ mouse, Heart &ndash 1ml/ 2 mice, 2ml/ 3 mice).
  2. Transfer to a 5mL polystyrene round bottom tube. Add Anti-mouse PECAM-1 Dynabeads (prepared in Section A) to cell suspension at 15 ul of beads / mL of cells suspension.
  3. Incubate on a rotator, RT, 10 min.
  4. Mount tube in a magnetic separator (e.g., Dynal MPC-S) and leave for 1-2 min.
  5. Remove supernatant. The beads + cells should look somewhat foamy if there is good yield of positive selection.
  6. Remove tube from magnetic separator and resuspend beads + cells in 3 mL Medium by vigorous trituration. Unless you are vigorous you will get many contaminating cells. (More relevent for the MLECs)
  7. Mount in magnetic separator for 1-2 min.
  8. Repeat steps 5-7 until the supernatant is clear (usually 4-5 washes).
  9. Resuspend in Growth Medium and plate beads + cells in one gelatin-coated T75 flask for lungs and one gelatin coated T75 flask for heart.
  10. The next day, rinse flask three times with isolation media to remove any loosely adherent cells.

  1. Feed with Growth Medium on Monday, Wednesday and Friday.
  2. Let cells grow until approaching confluence at 5-9 days after plating. You should see nests of &ldquocobblestone&rdquo EC comprising 10-90% cells. Other mesenchymal cells are notable for the way they pile up and don&rsquot form monolayers.
  3. Detach cells with Trypsin: EDTA (0.05%:0.5M) by rinsing 3x with Ca/Mg-free buffer, and then leave new Trypsin:EDTA on only as long as needed to achieve a suspension of cells.
  4. Add 10mL of isolation media to inactivate Trypsin.
  5. Transfer cell suspension to a 15 mL tube and spin down at 1300 for 8 min.
  6. Remove supernatant and resuspend cell pellet in 2 mL of DPBS-.
  7. Add ICAM-2 coated SAR Dynabeads to the cell suspension (15 ul/ml). Incubate for 10 min. RT.
  8. Place in magnet and leave for 1-2 min. Remove supernatant.
  9. Wash gently 2x in 3 mL media.
  10. After final wash resuspend in 10 mL MEC media and plate in gelatin coated T75. (Split at a ratio of 1:2 if the cell recovery is good).

  1. Feed with Growth Medium on Monday, Wednesday and Friday.
  2. Split cells 1/3 with Trypsin:EDTA when they reach 75-100% confluence.
  3. Replate endothelial cells on a gelatin-coated T75. It is important to maintain high density plating to avoid large numbers of senescent cells.


Cell culture splitting questions--why do you centrifuge the cells after neutralizing trypsin? And having issues with new lab techniques. is it just me?

Hi r/biology, I'm new here. I just started a research assistant position at a basic science lab on cells and of course, it's tough feeling useless in the beginning b/c all the research residents & techs are busy with either learning new techniques (blots, PCRs, etc) or busy w/their own projects.

My biggest question though is in cell splitting--why do you centrifuge after trypsinizing cells? I have always learned in my previous lab position, that after trypsinization of cells, neutralizing tryspin w/media, you have to centrifuge it for like 4 mins or so. Then you discard the supernatant, remix the pellet into a new 10mL of media and then distribute into plates. The lab here doesn't centrifuge they just pipette up and down for 5 mins and then they just shoot the mix into the plates.

As for me, previously, I worked 2 years in a basic science stem cell lab 3 years ago, and the only thing I did really well was cell splitting/plating/maintaining cell cultures. My old supervisor was super anal about being sterile and clean and wiping everything down w/alcohol, always wearing gloves, always dispose things properly, whether you were pipetting media or something really biohazardous. Especially in cell culture techniques.

At my new lab, besides feeling useless, I noticed there are major differences in how they carry out experiments. For example, when they pipette in the hood, they discard the tips randomly into some corner of the hood instead of into some 50 mL tube (how I was taught) or a tips jar. Or when they remove the old media from a plate, they don't use a vacuum + pasteur pipette (burned to be sterile), but pipette instead. They don't spray their micropipettes w/EtOH as often when they move things in and out of the hood. I'm not sure if I'm just undergoing adjustment issues or someting..

It's frustrating because I'm the lowest person in the lab, I have no skills beyond cell culturing but I dont want to be put in charge of other people's cells yet. And they (understandingly) don't believe me when I said I used to centrifuge cells. There's more things but I feel that each time I ask such things, it's a bit annoying to them. Like "oh well this is the shortcut" and I used to be drilled to do it in a certain manner by my previous lab manager 2.5 yrs ago who was extremely skilled.


Background

Clonogenic and MTT assays are well-known tests for evaluation of chemoradiation studies and radiosensitivity [1–4]. Clonogenic assays are commonly used to investigate survival of irradiated cancer cells, whereas MTT assays are well known to study chemosensitivity [5] or toxicity [6] of drugs in human tumor cell lines. The assay is less common to study survival of cancer cells after irradiation, in particular when the MTT assay is performed for studying proliferation of treated cells.

The aim of this study is to compare the well-established clonogenic assay with an adapted version of the MTT proliferation assay to overcome limitations such as long duration of experiment, low sample throughput, limited error level and time-consuming counting of clones.

The MTT assay is based on the formation of dark-colored formazan dye by reduction of the tetrazolium salt MTT by metabolically active cells [7]. After some incubation, the water-insoluble formazan dye forms crystals, which can be dissolved in an organic solvent and the amount can be determined semi-automatically using a microplate reader. Absorbance readings are related to the number of cells [8] therefore providing the possibility to use the MTT assay as a proliferation assay to assess cell growth after irradiation. In the present study our adapted version of the MTT assay is compared to the clonogenic assay in order to open the possibility of replacing one by the other.

In the literature, several studies on comparability of MTT and clonogenic assay can be found. There, the MTT assay is done as a single-point assay after a defined time following treatment. In this case, much information about the growth behavior of the cells is lost (doubling time, lag phase, growth behavior etc.). In contrast, with our multiple MTT assay, we collect all those data and use them for more detailed interpretation.


Discussion

In this manuscript, we outline a protocol to generate and propagate prostate spheres (prostatospheres) from prostate tissues and human and murine PCa cell lines. This assay depends on the ability of stem cells to survive in a 3D culture matrix (for example, Matrigel™) in low or serum-free medium while differentiated cells grow in adherent monolayer cultures and FBS-containing medium. Primary plating of single-cells suspension in a 3D matrix doesn't confirm the existence of an enriched stem cell sub-population, which is characterized by its self-renewal ability and differentiation potential. Multiple and serial propagations of the spheres are needed to confirm the existence of CSCs within a tumor or a cell line, dependent on finding a relatively constant SFU/SFE within each generation.

The issue of self-renewal represents a central feature of CSCs and is tightly associated with their pathologic ability to regenerate tumors after treatment. It represents the ability of these cells to reproduce indefinitely, while maintaining its multipotent ability to differentiate. 45 discussed this issue in terms of propagation and self-renewal of neuronal adult stem cells, cultured as neurospheres in suspension. (45) They properly argue that non-stem progenitor cells retain the ability of self-renewal and can indeed propagate into secondary or tertiary spheres (G2 and G3, respectively). Theoretically, prostate stem cells shall maintain self-renewal ability indefinitely. Due to technical limitations faced in vitro, propagating prostate cell-derived spheres over five generations (or more) can be practical to isolate and propagate prostate CSCs the sphere-forming ability of progenitor cells decreases subsequently, while that of prostate CSCs is maintained.

The clonal origin of the formed spheres is yet another important aspect of this assay. Previous work showed the tendency of neurospheres to coalesce and merge when cultured in suspension. In order to generate clonal colonies using cells grown in suspension to culture neurospheres, deposition of a single cell per well presented the golden criteria to reach that goal (39). Previous studies have successfully cultured prostatospheres in serum-free suspension culture, starting from PCa cell lines (51, 52). However, this issue hasn't been properly addressed. The protocol proposed herein suggests growing of cells in semi-solid matrix, to avoid their migration and aggregation, and overcome the technical burden of culturing non-adherent spheres starting from single cell per well. Discrete spheres were grown and monitored starting from a single cell suspension (Video S1). Matrigel™ provides an enriched matrix that harbors different glycoproteins and growth factors seen in the basement membrane, including collagen IV, laminin, and Fibroblast Growth Factor it provides a semi-solid matrix able of simulating the rich extracellular medium of cells in vivo (53). The use of Matrigel™ has been further implicated in long term organoids culture, another 3D-culturing modality that closely mirrors the in vivo prostate of murine and human derived cells (54). Moreover, Matrigel™ has been used to facilitate the establishment of tumorigenic Xenografts derived from human cell lines in immunocompromised mice (53).

A previous study found that the 3D solid spheres of mammary glands, cultured in Matrigel™, could be used for repopulating gland-free mammary fat pads in mice reaching an engraftment frequency of 71% (55). This means that the spheres formed can be used for tissue transplants and that this assay could be further developed in the future to be applicable on human tissue grafts. Interestingly, it was also found that the sphere assay could be used to compare the cellular mechanisms of normal and malignant cells through deciphering active pathways and differentiation patterns. It was found that the spheres derived from normal lung tissue, cultured in Matrigel™, were able to form a lumen while the spheres derived from cell lines and tumor biopsies of lung cancer were not (56), showing a potential use of the sphere-formation assay to compare the underlying cellular processes governing CSCs and those in normal tissue stem cells, and their pathophysiological implications on progression of the disease. Another study, by Hur et al. found that hematospheres could be isolated from blood and cultured in Matrigel™ to allow the formation of a network of vessels (57), proposing potential therapeutic implications to treat coronary artery occlusions or atherosclerotic arteries, by vessel replacement therapies.

Because the use of SFA has been recently implemented on assessing stem cell-like properties in tumors, there has not been much focus on drug treatments targeting these self-renewing cells. However, there have been various studies that focused on testing particular drugs on generated spheres. SFA was used, along with different assays, to show that Nigericin, an antibiotic that suppresses Golgi function in eukaryotic cells, suppresses colorectal cancer metastasis by inhibiting epithelial-mesenchymal transition (EMT) (58). The drug Metformin was shown to inhibit the growth of thyroid carcinoma cells, suppress the self-renewing property of the thyroid CSCs and act as an enhancing supplement to chemotherapeutic agents (43). Furthermore, Eckol has been proven to suppress the maintenance of stem cell properties and malignancies in glioma stem-like cells, hence suggesting a reduced chance of cancer recurrence (42). Also, spheres from esophageal tumor origin have been found to involve stem-like cells with elevated aldehyde dehydrogenase activity (59). Berberis libanotica Ehrenb (BLE) extracts as well were shown to have high therapeutic potential in targeting PCa and eradicating the self-renewal ability of highly resistant CSCs (35). All the mentioned examples have used SFA to assess the effect of drugs on the CSCs or stem cell-like populations in tumors these studies primarily relied on two main criteria in their assessment: average volume of the generated spheres and the average number of sphere-forming units (SFU). Yet, most studies focus on testing particular drugs on generated spheres for single generation, without assessing the effect on spheres propagated for several generations, which possess enriched stem cell/progenitor properties.

To emphasize the importance of the use of SFA in stem cell and cancer research, we have put together a detailed protocol of how to conduct a semisolid Matrigel™-based sphere-formation assay in the absence or presence of drugs, consecutively or alternatively, in the context of PCa stem cells. The advantage of using this sphere-formation in vitro assay is to isolate, propagate, purify, and amplify specific populations of prostatic normal and CSCs. It enables studying stem cells at different stages of their formation (at different generations) and detecting markers of their signaling pathways. It also solely depends on the functional intrinsic property of stem cells in forming a complex structure in a 3D environment, namely self-renewal ability and differentiation potential.


Genomics Pioneer Craig Venter Envisions Future of Synthetic Life

NEW YORK — Life is a DNA software system, genome scientist Craig Venter told a packed auditorium here at the American Museum of Natural History Monday night (Oct. 21). In his talk, Venter offered a longsighted view of the creation and digitization of synthetic life.

Creating synthetic life is just a crowning achievement of Venter's career and the evolution of the field of biology. In 2000, Venter led of one of the two teams that sequenced the human genome, the blueprint for life. Then in 2010, his team transplanted man-made DNA into a bacterial cell to create the first synthetic organism.

To create a synthetic cell, Venter said, he and his colleagues had to find a way to write the DNA software and boot it up. And this technology opened up a host of practical applications, he explains in his new book "Life at the Speed of Light" (Viking Adult, 2013), in which Venter tells the story of these milestones and speculates on the future of biology in the digital age. [Unraveling the Human Genome: 6 Molecular Milestones]

Biological teleportation

His ideas only get more unusual from there. What if, Venter speculated, one could send a genome across the solar system at the speed of light, and reconstitute it on the other side? For example, if a rover discovered life on Mars, it could sequence the life-form's DNA and beam the code back to Earth, where scientists could rebuild the organism.

Of course, Venter was talking about simple life-forms such as bacteria. "We're not ready to beam humans across the universe anytime soon," he said.

But the reality is still impressive. The ability to synthesize life from its DNA alone could vastly accelerate vaccine production, Venter said. Scientists could sequence an emerging flu virus anywhere in the world, and send that sequence over the Internet to pharmaceutical companies that could develop a vaccine for it. Ultimately, he said, people may be able to download genetic sequences to a machine that produces vaccines in their own homes.

Venter and his colleagues have laid the foundations for these developments by developing the tools needed to construct living cells.

Synthesizing life

The first step, Venter explained, was making software that could build its own hardware. His team created a synthetic bacteriophage, a virus that infects bacteria, and injected that into E. coli bacteria cells. The cells incorporated the synthetic DNA into their genomes, and they started assembling bacteriophages. [5 Crazy Technologies That Are Revolutionizing Biotech]

Venter's next project was ambitious: His team modified a chromosome from the bacterium Mycoplasma mycoides and inserted it into the cell of the bacterium Mycoplasma capricolum. To do that, his team had to develop sophisticated new genetic techniques.

Once inserted into the host, M. mycoides' DNA started making instructions for enzymes that chewed up the host bacterium's genome. The result? "We transplanted the genome from one cell into another species, and in the process of doing that, converted the one species into the other," Venter said.

The final step was to piece together an entire bacterial chromosome and put that into a cell where it would replicate — no easy feat. To do that, Venter and his team created big chunks of bacterial DNA and assembled these inside a yeast cell. After several roadblocks and years of trial-and-error, the scientists produced the first synthetic cell in 2010.

The synthetic genome contained a "watermark" sequence that included the names of the scientists who worked on it. It also included quotes by physicists Richard Feynman and Robert Oppenheimer, and this quote by writer James Joyce: "To live, to err, to fall, to triumph, to recreate life out of life."

Playing God?

Insofar as the team created an organism capable of thriving and self-replicating, Venter and his colleagues had created life.

"In the restricted sense that we had shown with this experiment how God was unnecessary for the creation of new life, I suppose that we were," Venter writes in his new book.

But for Venter, synthesizing life is merely the logical outcome of years of genetic tinkering.

Modern biology was born, Venter believes, when Austrian physicist Erwin Schrödinger gave a series of lectures entitled, "What is Life?" in Dublin in 1943. Schrödinger proposed that chromosomes were a kind of "code script," which might be as simple as Morse code.

In 1944, three Canadian and American scientists — Oswald Avery, Colin MacLeod and Maclyn McCarty — performed an experiment that proved DNA, and not proteins, was the hereditary material of cells. And in 1953, American biologist James Watson and his British colleague Francis Crick discovered the structure of DNA, building on work by Rosalind Franklin and Maurice Wilkins.

The 1960s and 1970s witnessed huge advances in the understanding of DNA and recombinant DNA technology. Building on these foundations, Venter's group and the publicly funded Human Genome Project produced the first draft sequence of the human genome in 2000.


Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines

Pseudomonas aeruginosa is an opportunistic pathogen of plants and animals, which produces virulence factors in order to infect or colonize its eukaryotic hosts. Cyclodipeptides (CDPs) produced by P. aeruginosa exhibit cytotoxic properties toward human tumor cells. In this study, we evaluated the effect of a CDP mix, comprised of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) that were isolated from P. aeruginosa, on two human cancer cell lines. Our results demonstrated that the CDP mix promoted cell death in cultures of the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.53 and 0.66 mg/mL, for HeLa and Caco-2 cells, respectively. Flow cytometric analysis, using annexin V and propidium iodide as apoptosis and necrosis indicators, respectively, clearly showed that HeLa and Caco-2 cells exhibited apoptotic characteristics when treated with the CDP mix at a concentration <0.001 mg/mL. IC50 values for apoptotic cells in HeLa and Caco-2 cells were 6.5 × 10 −5 and 1.8 × 10 −4 mg/mL, respectively. Our results indicate that an apoptotic pathway is involved in the inhibition of cell proliferation caused by the P. aeruginosa CDP mix.

1. Introduction

Pseudomonas aeruginosa colonizes many biological environments, such as soil, plants, and animal tissue, being an important pathogen involved in opportunistic infections in humans [1] and a major cause of nosocomial infections [2]. Several mechanisms for driving infection in the host have been attributed to P. aeruginosa, and, among these, the production of toxins, adhesins, pyocyanin, and other virulence factors plays an important role in infecting different hosts, from plants to animals [3, 4]. P. aeruginosa produces and senses N-acyl-L-homoserine lactones (AHLs) for cell-to-cell communication via a regulatory mechanism known as quorum sensing (QS), which links the perception of bacterial cell density to gene expression. QS coordinates many physiological processes, such as symbiosis, production of virulence factors, resistance to oxidative stress, antibiotic resistance, motility, biofilm formation, and the progression of P. aeruginosa infection in animals [5, 6].

The cyclodipeptides (CDPs) cyclo(D-Ala-L-Val) and cyclo(L-Pro-L-Tyr) have been identified in P. aeruginosa cultures, which led to the proposition that CDPs have the ability to inhibit the activity of regulatory LuxR-type proteins that are involved in AHL-dependent QS signaling. This in turn led to the proposition that CDPs and their derivatives, the diketopiperazines (DKPs), represent a new class of QS signals and that they could potentially act as interkingdom signals. However, the mechanism of action and physiological relevance of CDPs are poorly understood [7, 8].

DKPs are cyclized molecules comprising two amino acids bound by two peptide bonds they are produced by a wide range of organisms, from bacteria to fungi and animals (Figure 1(a)) [9, 10]. DKPs belong to the nonribosomal peptides that are synthesized in microorganisms by a multifunctional assembly of enzymes known as nonribosomal peptide synthases [10] and by CDP synthases, another kind of enzymes that utilizes aminoacylated transfer RNAs as substrates instead of free amino acids [11].

CDPs are structurally diverse, and they have been implicated in multiple functions the CDPs cyclo(D-Ala-L-Val) and cyclo(L-Pro-L-Tyr) have been identified as a new class of QS autoinducers in Pseudomonas strains, based on their ability to activate AHL-dependent biosensors [12–14]. The CDP cyclo(L-Phe-L-Pro) isolated from Lactobacillus plantarum exhibited an antifungal effect against Fusarium sporotrichioides and Aspergillus fumigatus [15], while the CDPs cyclo(L-Leu-L-Pro), cyclo(L-Phe-L-Pro), cyclo(L-Val-L-Pro), cyclo(L-Trp-L-Pro), and cyclo(L-Leu-L-Val) isolated from the deep-sea bacterium Streptomyces fungicidicus showed antifouling effects [16]. Moreover, synthetic CDPs such as cyclo(Phe-Pro) induced apoptosis in the HT-29 colon cancer cell line [17], and cyclo(L-Cys-L-Leu) exhibited potential for scavenging free radicals [18]. Recently, it was reported that P. aeruginosa is capable of interacting with the plant Arabidopsis thaliana via the secretion of CDPs such as cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe), appearing to mimic the biological role of auxin, a natural plant hormone [12] (Figure 1(b)). In Staphylococcus aureus, the aureusimines A/B, comprised of the CDP cyclo(L-Val-L-Tyr) and cyclo(L-Val-L-Phe), respectively, are involved in the regulation of bacterial virulence factors in a murine host [19] similarly, the CDP cyclo(L-Phe-L-Pro) in Vibrio cholerae, V. parahaemolyticus, and V. harveyi is involved in controlling the expression of genes that are important in pathogenicity [20]. Moreover, it was reported that CDPs and DKPs may induce cell death in several cancer cell lines [21], by affecting biological processes such as microtubule polymerization for example, cyclo(D-Tyr-D-Phe), isolated from Bacillus species, induced apoptosis via caspase-3 activation in the A549 pulmonary adenocarcinoma cell line [22]. In addition, it was reported that the CDPs cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) isolated from Lactobacillus exhibited antiviral activity against the influenza A (H3N2) virus [23].

Although, in the context of bacteria-mammalian interaction, it has been suggested that CDPs could play an important role in bacterial pathogenesis, bacteria-host signaling, or mammalian cell growth, the mechanisms involved are unknown. Therefore, in this study, we focused on investigating the cellular effect of CDPs produced from P. aeruginosa strain PAO1, a pathogenic bacterium in humans that is capable of secreting the CDPs, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) into the culture medium (Figure 1(b)). The biological effects of these CDPs on the growth and/or pathogenesis of mammalian cells remain unknown the P. aeruginosa CDPs could be involved in bacterial host colonization phenomena during disease episodes, where antiproliferative or anti-immune properties of these compounds could affect the host organism. In this regard, we employed the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines as host models in this study.

2. Materials and Methods

2.1. Chemicals and Reagents

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotic antimycotic solution (100X) penicillin, streptomycin, and amphotericin B were purchased from Sigma-Aldrich Co. 4,6-diamidino-2-phenylindole (DAPI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. Alexa Fluor 488 annexin V and the PI/dead cell apoptosis kit were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Tissue-culture plastic ware was acquired from Corning (Tewksbury, MA, USA).

The P. aeruginosa CDP mix was characterized as described previously [12]. Briefly, the P. aeruginosa WT strain was placed in 100 mL of Luria Bertani (LB) medium and incubated for 24 h at 30°C for bacterial growth. Cell-free supernatants were prepared by centrifugation (10,000 ×g, 25°C for 10 min). The resulting supernatant was extracted twice with ethyl acetate supplied with acetic acid (0.1 mL/L). The extracts were evaporated to dryness using a rotavapor at 60°C (Buchi Co., Lawil, Switzerland). The residue was solubilized in methanol : acetonitrile (1 : 1) and analyzed by GC-MS as described [12]. The CDP mix is constituted by the cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) in a 1 : 1 : 1 molar ratio. For dose-response assays, the CDP mix was evaporated to dryness, weighed out, and dissolved with DMSO to prepare a 100 mg/mL concentration as stock solution.

2.2. Cell Line Growth

The human cancer cell lines HeLa and Caco-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell procedures were performed under class II biological safety cabinets. Cells were cultured in DMEM supplemented with 10% (v/v) FBS (complete medium) and 1% antibiotic (10,000 units of penicillin, 10 mg streptomycin, and 25 μg of amphotericin B per mL) solution. The cultures were fed twice a week and maintained at 37°C under 80% humidity and incubated in an atmosphere of 5% CO2. HeLa and Caco-2 cells were collected by trypsinization using trypsin/EDTA buffered solution for 5 min at room temperature, followed by the addition of 5 mL of serum-enriched medium (CM) to stop trypsin action. After trypsinization the cells were collected and washed with CM. Finally, cells were counted in a hemocytometer chamber and incubated in fresh CM media.

2.3. Cell Viability Assay

Cell viability was determined by the MTT colorimetric method using thiazolyl blue tetrazolium bromide (Sigma-Aldrich Co). Briefly, HeLa and Caco-2 cells were seeded in 96-well flat-bottomed plates at a density of 3 × 10 4 cells per well in 200 μL of CM and incubated for 24 h at 37°C as described above. Then, the medium was removed and replaced with new CM or serum-free medium (SS). Then, cells were incubated with CDP mix solution at indicated concentration. Cells were incubated for another 24 h at 37°C. To determine cell viability, 10 μL of 5 mg MTT per mL in PBS was added to each well and incubated for 4 h at 37°C. Finally, 100 μL of 2-propanol/1 M HCl (19 : 1 v/v) was added to dissolve the formazan crystals. Absorbance measurements were conducted utilizing a microplate spectrophotometer (IMarK Microplate Reader, BIO-RAD, Hercules, CA, USA) at 595 nm.

2.4. Necrosis and Apoptosis Assay

HeLa and Caco-2 cell lines were seeded in 96-well flat-bottomed plates at a density of 3 × 10 4 cells per well in 200 μL of CM and incubated for 24 h at 37°C. Then, cells were synchronized with SS medium for 12 h at 37°C and were incubated with different concentrations of CDPs mixture. DMSO was used as control at same concentration used to dissolve the CDP mix. To determinate the apoptotic effect, cells were collected by centrifugation at 2,000 ×g for 10 min. The pellet was suspended in 20 μL of SS medium and treated with annexin V and propidium iodide (PI) (Dead Cell Apoptosis Kit Molecular Probes, Invitrogen Life Technologies, Carlsbad, CA, USA) following the indications recommended by the manufacturer. Fluorescence was immediately quantified by flow cytometry (FC) using a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). The populations of cells for each of the treatments were gated in forward scatter and side scatter dot plots to eliminate cell debris. Populations corresponding to auto- or basal-fluorescence were located in the left quadrant and cells with emission of fluorescence increasing at least one log unit value were located in the right quadrant of the dot plots. In addition, the percentage of fluorescent cells (PFC) and median fluorescence intensity (FI) were determined in monoparametric histograms of fluorescence emission obtained from the dot plots and labeled as PFC and as relative units of fluorescence. The equipment was calibrated using Spherotech 8-peak (FL1–FL3) and 6-peak (FL-4) validation beads (BD Accuri, San Jose, CA, USA). For apoptosis and necrosis assays, fluorescence for annexin V in emission fluorescence channel FL1 at 495/519 nm and for propidium iodide in the FL2 channel at 535/617 nm was monitored. A minimum of 20,000 cellular events were analyzed.

2.5. Cell Image Captures

HeLa and Caco-2 cells were seeded in 12-well flat-bottomed plates with sterile-covered round objects covered with collagenase at a density of 1 × 10 4 cells per well with one mL of CM and incubated for 24 h at 37°C. Cells were incubated with serum-free medium (SS) for 12 h at 37°C and an atmosphere of 5% CO2 and incubated with different concentrations of the CDP mix. After 12 h of treatment, the cells were washed with PBS. Cells were fixed with paraformaldehyde (PFA at 4%) for 10 min on ice. Then, cells were incubated with DAPI (1 : 1,000) for 10 min at room temperature. Finally, cells were washed with PBS, and the cover glass was removed and placed into a holder with a drop of PBS and glycerol 1 : 1. Cultured cells were photographed using an inverted phase-contrast microscope (Carl-Zeiss HB0-50, San Diego, CA, USA) equipped with an AxioCam/Cc1 digital camera. Cultures of HeLa and Caco-2 cells were grown in CM and incubated with DAPI and visualized using a confocal microscope (Olympus FV1000, Center Valley, PA, USA). The cells were observed by fluorescence emission between 405 and 505 nm.


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Each type of cell or cell line responds to Trypsin-EDTA for Primary Cells in a unique manner. For optimum results, continuously observe the cells during the dissociation process to prevent damage. For cell-specific information, please refer to the product sheet supplied with the cells or cell line.

  1. Bring the DPBS, the Trypsin-EDTA for Primary Cells, and the Trypsin Neutralizing Solution to room temperature before use. Warm the complete growth medium to 37°C prior to use with the cells.
  2. For each flask, carefully aspirate the spent media without disturbing the monolayer. If the cell culture medium contains serum, each flask should be rinsed with DPBS twice prior to adding the Trypsin-EDTA for Primary Cells.
  3. Using 1 to 2 mL for every 25 cm2, add the appropriate volume of trypsin-EDTA solution to each flask (e.g., each T-25 flask would be dissociated with 1 to 2 mL trypsin-EDTA).
  4. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer do not aspirate to dryness.
  5. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within about 3 to 6 minutes), remove the flask from the microscope and gently tap the culture flask from several sides to promote detachment of the cells from the flask. Do not over-trypsinize as this will damage the cells.
    1. Some strongly adherent cell types, such as keratinocytes, may take much longer and may require trypsinization at 37°C.
    2. Some cell types may require more vigorous tapping.
    1. Do not over centrifuge cells as this may cause cell damage.
    2. After centrifugation, the cells should form a clean loose pellet.

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