Science method

3D Cell Culture - Science method

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Hi! I am trying to prepare hydroxyapatite scaffold samples for SEM imaging of cell growth. I have the Karnovsky's fixative kit but the procedure provided in the tech sheet (attached) is not sufficient for my applications. First, does anyone have a standard protocol for this SEM fixation using Karnovsky's fixative kit? Second, do I need to do the post-fix using OsO4 or is there an alternative method to the post-fix mentioned in the tech sheet? Can I do the fixation procedure without it, followed by the graded ethanol dehydration or will it have a negative impact on my sample preparation?
I would really appreciate any help answering this question. Thanks!
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If you have a cells monolayer, 30 min is a good time. If you have something like a tissue developing, with a lot of collagen, then you need 1 hr. HA is soluble in water (very slow, but still...). So if you culture started generate small centers of mineralization, you do not want to keep it too long (days, weeks) in water solutions. From the other side prolonged storage in desiccator can lead to fungus growth. Some desiccators are badly infested with fungus and need through cleaning and disinfection. From my opinion the best way to store specimens is when their preparation is complete, i.e. they are dehydrated and coated with conductive coating.
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I am culturing MSCs on 3D-printed hydroxyapatite scaffolds. We need to detach cells from the 3D to analyze/quantify overall DNA content using Quant-iT PicoGreen dsDNA Reagents and Kit. However, these protocols have not been tested or adapted for complex 3D cultures. We aren't sure what the best method would be to detach & cells from the 3D scaffold and lyse the cells. We also need a technique to verify that our adapted method is effective. I'm interested in hearing what techniques/protocols others are using or any recommendations. Thanks!
Our currently drafted protocol, which is subject to change, involves the following steps:
1. Get DNA standard lysates using PureLink Genomic DNA Kit of cells prior to seeding.
2. After culturing cells seeded on 3D scaffolds for _____ days, at different timepoints, transfer the scaffolds to new wells in 24-well plates so that cells adhered to the wells are excluded.
3. Add TrypLE to the scaffold wells and incubate them, on an oscillating shaker to promote detachment, for 20 minutes.
4. Collect the trypsinized cells and transfer to centrifuge tubes.
5. Add TrypLE to the scaffold wells again and incubate for 10 minutes. Then, repeat collection & transfer of trypsinized cells.
6. Do a 2x rinse using trypLE to try to "knock off" remaining cells and collect as many cells as as possible from the matrix. Repeat until the TrypLE collected is clear, not turbid, hinting that there are little cells remaining in trypsinized suspension.
7. Centrifuge the trypsinized cells to isolate the cell pellet.
8. Resuspend cells in PBS.
9. Follow the protocol in the PureLink Genomic DNA kit to prepare unknown content of DNA in the cell lysates.
10. Follow the Quant-iT PicoGreen Kit protocol to complete the reactions & quantify dsDNA in the samples.
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Benjamin Fournier I was considering doing so but I wasn’t sure if cells should/could be lysed straight off the scaffold without detaching first. I am concerned that I won’t extract all of the DNA if cells are trapped on the internal area of the scaffold. I will have to make sure that each internal pore/cavity is exposed to the lysis buffer but I believe it should work. Have you ever tried to lyse cells directly from a scaffold? Thanks!
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Can anyone please suggest a cheap hydrogel brand available to buy for 3D cell culture?
Or if someone can share a recipe to prepare homemade hydrogel (for 3D cell culture), that would be fantastic.
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If you want to grow spheroids, you can use 0.6% agarose-coated well for cell growth. Jarshad Jas
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I am a green hand in 3D cell culture. I used U-shape bottom 96-well plate with a hydrophobic coating for 3D cancer cell culture, and I found it very difficult to remove or change media without aspirating the spheroid. Are there any tips for that? Thanks!
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I wonder what is your goal in completely removing the media. However, if you only wish to renew/refresh the old medium, I suggest you to do it by half the total volume, e.g., 100 uL removal from a total of 200 uL. Aspirate the said amount slowly without touching the U bottom. You can tilt the plate to the side, so your pipette tip is aiming to the wall. Do the medium renewal more often if you worry about not providing optimal condition for your spheroid.
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Hi I'm looking at making collagen hydrogel with different stiffness for 3D cell culture . I am going through literature as I wait for responses here but would be interested in recipes that have been used before. Also, any tips on successful preparation? This is a new technique for me.
Thanks
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We recently blogged about a study making hydrogels with varied, tunable stiffnesses. The link is below and the original paper can be found there as well. The authors examined the effectiveness of SV-hydrogels in reprogramming and manufacturing hMSCs with designed biomechanical properties for improved therapeutic potential.
The tunable hydrogels were from Cellendes and available in U.S. from Ilex Life Sciences.
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Hello. So I had seeded some 48-well plates with 70,000 cells/well (human osteoprogenitor) on a calcium phosphate substrate scaffold.
After 24 hours I wanted to study the overall attachment so basically I incubated I removed the scaffolds from the wells and placed in to a new well plate.
I washed with PBS and then incubated in a trypsin 1x solution for 30 minutes to allow for the cells to deattach from the scaffold.
I then neutralized with media and centrifuged the solution to form the smallest dot sized pellet.
I removed the solution and reconstituted in 1 mL of media and then using 10 uL of the solution, mixed it with 10uL of trypan blue.
Transferred 10 uL to side A and side B of a chambress counting slide and used the corresponding automated cell counter by life technologies to count the cells.
I somehow got a bigger number - for example 4.5x10^5 cells alive/ mL
The time point was only 24 hours so it’s not possible that the cells divided that quickly and multiplex in that number so fast.
Can someone please help me understand the principles behind automated cell counting because I believe the machine maybe possibly multiplying by a factor to estimate the number of cells? Please help because clearly the machine won’t count cells which aren’t there, I just don’t understand why it’s spitting out such larger numbers.
Please it’s my last experiment of my thesis and I just need a little help please.
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Dear researcher
See the following papers method
Cell viability analysis using trypan blue: manual and automated methods
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Hello,
I am conducting CRISPR experiments on some cell lines. At the end of the experiment, I harvest the DNA, amplify with PCR the region of interest and I send it to a company that does Sanger Sequencing in order to be able to identify the presence of insertions and deletions. However, in my last set of experiments I am having trouble in achieving good sequencing readouts of the controls. These cells undergo DNA extraction with a quiagen tissue extraction kit (as they are in 3d cell culture with matrigel, and this is the standard extraction method used in the lab), then they are amplified with a normal PCR and PCR purification is done by column purification with the nucleospin PCR cleanup kit from macherey-nagel (last elution step in ddH20, repeated two times). The concentrations of the DNA I obtain is in the range required from the Sanger sequencing provider. Do you have any advice on how I could increase the DNA purity? Or is there something else that I am mistaking? Thanks a lot!!
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Thank you for the files Sofia Visconti . I will get back to you later today but meanwhile are you purifying away the pcr primers from the pcr product before sequencing? Are you using exo-sap degradation of the primers or is the sequencing company removing the primers for you please?
Thank you Paul
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I am working on developing 3D cell cultures of tumor cells and I noticed that there are different methods, like liquid overlay, air interface culture, hanging drop technique, and scaffold-mediated approaches.
As for the liquid overlay approach, the general idea is to create a surface that cells cannot adhere onto. I am curious about whether I can use matrigel to replace agar/agarose or HEMA for the development of 3D cell culture?
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Hi Weizhong,
One alternative is plant nanocellulose hydrogels (GrowDex) which are well-defined and animal-free matrices for 3D cell culture. Cells can grow in all three dimensions in the hydrogel where the cellulose nanofibers form the network and provide physical support for the cells. This is one step closer to in vivo situation, compared to suspension cultures on ultra low attachment wells where is no fiber network. Please check a couple of publications with cancer cell data:
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Hello, my work involves growing cells in the matrigel and for recovering cells from matrigel i usually use cell recovery solution from corning. As now i am curious to know if theres an alternate to recover the cells from matrigel. The mechnical pipetting doesnt work well for me. Please do suggest if theres an alternate methodbt
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Hi Madhura,
If you don't want to stress the cells with ice cold culture media, there is a possiblity to use another 3D culture matrix, such as plant cellulose -derived GrowDex. Cells can be isolated from GrowDex with simple and safe method with GrowDase enzymer treatment. The enzyme does not affect the cells at all, retaining e.g. pluripotency of iSPCs and ESCs. Also cell surface proteins are intact. Please take a look of this article where they used GrowDex to 3D culture of iPSCs and ESCs:
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Hello Scientists,
In our study in SERS, our cells either break down.Not enough peaks from non-lysed cells.The intensity values of the incoming peaks are low and their number is low.We used many surfaces but the situation is the same.
What could be the problem?
Thanks in advance
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Dear Nazli Öncer , SERS experiments are often plaged with lack of reproductivity and while some measurements can yield good spectra, some other do not show any signal at all or it is flooded by luminiscence. This is true with any sample you are analysing, from simple small molecules to large and complex proteins. Additionally, as with any anylitical technique, the sample prepraration is very important, specially when you are dealing with difficult matrices, such as cells or their whole composition if you break them. It is true that SERS has been applied successfully to identify cells, bacterias and viruses, but often taking advantage of some cell membrane to attach or atract some plasmonic nanoparticles to its surface or to bind the cell to an extended plasmonic substrate. Take for instance the case of SARS-CoV-2 virus, you could design an antibody that binds to the spike protein of the virus' corona, and that antibody bonded to the plasmonic nanoparticles or to the surface of your SERS substrate. This would bind to the virus and it will be closer to the plasmonic material, so that when the laser beam excite the localised plasmons, the virus (cell, etc) would feel the intense local field and will emit its characteristic spectrum.
SERS can work without these linkers as well, but anyway, you need to place your molecule of interest very very close to the plasmonic surface, otherwise the signal would be very low or null.
When you have a very complex sample with lots of different components it could be hard to get a good SERS response if any at all. Why? Well there are multiple reasons, for instance, imagine your sample is very concentrated, so that the sample material forms a thick shell around the nanoparticles or a thick film over the SERS substrate, light will hardly reach the plasmonic material and therore no excitation happens. Imagine that your molecule of interest is present in your sample in low concentration, you still have all the rest in the media, that matrix can also shadow the SERS response, those other materials coat the surface and few or none of your target molecules can reach it, so they wouldn´t feel the enhaced field and then the signal will be poor.
Another annoying effect of the matrix could be the luminiscence effects, so may be some of your target molecules are close enough to the plasmonic surface, giving a good signal, but also some matrix molecules are touching the surface and well they also can give their own enhanced Raman signal or well show intense luminiscence that floods the Raman signal of your sample. Also both effects can happen simultaneously (Raman and luminiscence from the matrix).
The linker (antibodies, functional groups, etc) are useful to selectively bind the target molecules to the substrate, and then rinse the SERS substrate to remove the matrix or a larger part of it, so that you get rid off these matrix interferences
When you are working with extended substrates, the place where you point the laser focus matters too. Often the sample is not evenly distributted in the surface and if you pointed over an area where there are few molecules of interest, the signal will be small, while if you shift a bit microns apart the laser focus you can find a strong response because there there is a right concentration of molecules close enough to hot spots.
How do you know this? The answer is generally trial and error. A way to improve this is by making Raman imaging, if available in your Raman spectrometer/microscope. The idea here is to scan a region of interest (ROI) over your substrate, looking for some band that you can assign to your molecule of interest. The image would show you a map of intesities, so you can see where the signal from your target molecule is higher and there perform further local analysis that will let you get a good Raman spectrum.
Of course your molecule must be Raman active. Laser wavelength, laser power, microscope objective are also paremeters to take into account. For instance, a low power would not retrieve a good signal, but too much power will burn your sample.
Recently the following review about SERS and cells was published:
More reviews or research papers can be easily searched through the RG search funtion or with any other searching engine.
Hope it helps. Good luck with your researh and my best wishes.
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I need to prepare a 0.04mg/ml collagen type IV solution in 50mM of Tris-HCL to incubate and coat some cells. Thus, I have been thinking and I guess that I bought an inadequate product to get my solution.
I have only 1 vial containing 1ml at 0.3mg/ml of collagen and I would intend to have more than 5ml of the final solution.
Would someone have some advice or another way to prepare this type of solution?
Best Regards
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Ok, thats no problem. I agree to Ellen's comment
or Ca x Va=Ce x Ve -rule
Ca=0,3 mg/ml (starting-conc.)
Va=1 ml (starting-volume)
Ce=0,04 mg/ml (end-conc.)
Ve= x ml (end-volume)
-> Ve = (Ca x Va)/ Ce
-> x ml = (0,3 x 1)/0,04 = 7,5 ml (end-volume)
This means
-> 1ml of your solution + 6,5 ml of your buffer results in 7,5 ml of your collagen solution with a conc. of 0,04 mg/ml
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We are trying to measure growth kinetics in 3D cell culture system. In the assay, we would like to take Cyquant reading from each well at Day0, 1, 2 and so on. Can we use the same wells to perform this kinetic assay?
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Hello!
The possibility to measure changes in proliferation in the same well is complex. Although this CyQUANT product does not need to lyse the cells, I understand that it is not possible to remove the probe that binds to the DNA.
While the intensity of the probe decays after 4 hours, you will never know for sure if those binding sites become available again.
Perhaps an alternative might be to tag the cell and get videos of a few hours that will allow you to evaluate its kinetics.
Hopefully you will find a solution!
Regards
Lucia
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Could anyone share the protocol for cell recovery from gelatin scaffolds without a substantial loss of cell number?
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Agreed with Henry E Young, well-explained. However, the 3D-cell culture here is not really clear, are you talking about pre-seeding on the gelatin surface or pre-mixed prior to forming 3D-scaffold, especially hydrogel form. Using an enzymatic approach thru collagenase might work but the time of incubation is really critical to avoid over-digestion that at last may cause cell deterioration..Best of luck!
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I am interested to acquire Z-stacks via brightfield microscopy of spheroids comprised of Head and Neck cancer cells. My objective is to acquire sphericity and volume on day 0 of the experiment (72h after seeding into ultra-low attachment 96-well plates), on day 1 (24h after treatment with drug of interest), day 2 (48h after treatment) and day 3 (72h after treatment). Due to several time points, it is important that the spheroids must not be fixed for sphericity and volume measurement. After some literature research I was not able to find a microscope model, where I can put my 96-well plate for acquiring z-stacks.
Maybe someone here has encountered a similar problem and could help me, I would be very thankful for any help in this matter. I already have found the software, which I would need for the measurement (arivis vision 4D) but still do not know, with which microscope model I should acquire the images. Is it even possible to acquire z-stacks spheroids without putting them on a slide?
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Hi Sam
I am not sure if you can successfully define the volume of a spheroid using brightfield imaging, I'd prefer fluorescence microscopy and - depending on the sample size required - with an optical sectioning capability.
For the small sample size I suppose Fahimeh's suggestion then works. For the larger ones were you keep the sample in mulitwells most high content screening setups which have confocal will then be suitable to image with the better imageing result in the lower part of the object towards the objective on the inverted setup (depending on size you might more or less of the total but after 72h you might only get a very small part of the control).
There are also solutions available providing very low phototoxicity based on light sheet microsopy, not sure if you can purchase them yet (e.g. oblique plane microscopy, SCAPE but also commercially available light sheet microscopy e.g. Zeiss) but you might need to establish a different sample mounting method. A commercially available solution for multiplexed 3d light sheet imaging has been developed by Luxendo now Bruker....
There is loads to think about and a lot depends on your assay size, the resolution you require and your financial resources. Don't take my suggestions as complete.
Best
Heiko
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I tried to make alginat-Ca hydrogel bead.
When I put the beads in pure water, they could be stable for a long time.
But when I put them in RPMI 1640 Medium, they became soft like slime and the beads would break into pieces.
Why does RPMI 1640 Medium break the alginate beads?
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I suspect the amino acids in the medium will compete for chelation of the calcium ions.
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If anyone has experience working with gelatin-based hydrogels for culturing 3D cell culture of mammary epithelial cells, could you please comment on synthesis of gelatin hydrogels ?
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Gelatin-based hydrogels are simple and easy to make on your own. I have synthesized methacrylated gelatin (chemical modification) and gelatin-based thin films (crosslinkers used). There are tonnes of research articles that are focused on creating these gelatin-based hydrogels. But if you don't want to go through the process of making your own, there are also a lot of companies that sell commercial gelma for research.
Having said that, here are some pointers to consider when working with GelMA. Mechanical properties of hydrogels can affect how your cells behave, so ensure you read literature to find out details on what percentage of gelma would be ideal for your experiments, what percentage of methacrylation the components have (higher the methacrylation amount means higher photo crosslinking and more stiffer gels). I focused more on GelMA here but similar principles apply for most of them.
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Hi All, I am working with A549 cell line and trying to culture spheroids using low attachment 96 well plates. So far I have attempted some different seeding densities from 2000 to 10,000 cells and can either form very large spheroids (700-900um), which are more compact and have a spherical defined shape, or alternatively smaller spheroids (still fairly big though around 500um) are less compact and not completely spherical. However for my experiment where I wish to add drug compounds (2D IC50 approx 1uM) I am not observing significant size/morphology change on the larger spheroids despite at least a 10uM concentration for 1 week. I am thinking possibly I can try to treat smaller spheroids for a more obvious visual change. Does anyone know how i might successfully make small compact spheroids (less than 500um) which are reproducible with this cell line? Thanks in advance for any help someone may be able to provide.
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Hi Sophie, I have worked with this cell line and also for spheroid formation in my Ph.D. thesis, So I suggested you control the cell number when you going to seed in 96 well, this in turn leads to control the size of the spheroid, however, if you use microfluidics or lab on chip systems you can generate a huge number of the spheroid in one chip and also control the size of the spheroid, one of the main factor in drug screening studies (larger than 400 um in size spheroid are not suitable as the cells in core zone can not access to nutrients and drug and also they are under hypoxia),
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I need to fix the 3D tumor spheroid that has been constructed using the low attachment plate method for confocal imaging. We are having problems with fixation without changing the sphere shape. Does anyone know a better method to overcome this issue?
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you could try to use a low-melting agarose.
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Hi,
I am currently working with osteoclast differentiation using the hanging drop technique. I found that droplets at the border have better spheroid formation, compared to the droplets in the middle of the petri dish.
Does anyone else have seen these results? And what might be the explanation between these differences?
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Simply, go where the most and best food is available. :-)
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I am going to start spheroid culture with Glioblastoma and Medulloblastoma cell lines. I read about including BIT admixture 100 supplement in several publications to support culture.
On the companies website I took the information that you can use this admixture to replace serum.
Has anybody experience with the supplement and Can explain to me what exactly the benefit of adding this would be? Or if I could just forget about it...
Many thanks,
Celine
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Dear Celine!
BIT Admixture - 100 is a complete medium formulated for optimal growth of tumor stem cells, e.g. glioblastoma stem cells and in cases where defined medium compositions are required such as colony forming assays or growth of hematopoietic progenitors. It contains essential components that help to replace serum.
If you have a good serum, you can simply use a medium serum without BIT supplement
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I have been working with 3d spheroids for a few months. I started with HCT-116 colon cancer cell line and have not had any problems, this cell forms tightly aggregated spheroids. However, I have not been able to form spheroids from HT-29 cell line. I've tried different agarose concentrations (1-3%) and different cell concentrations (800-10.000 per well) and this cell line won't form the spheroids, just cell clumps. Does anyone have any idea what could be happening? I've seen in many papers spheroids from HT-29 and theorically they are formed easily. I use the following protocol for the HCT-116:
96 well plate flat bottom coated with 50uL of 1.5% agarose. I plate 2000 cells and centrifuge 1000rpm for 5 minutes. Then, I incubate for 4 days untill the spheroids are formed.
Thanks in advance.
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Dear Gabriel,
my team and I have recently published a paper on tumor spheroids (https://www.frontiersin.org/articles/10.3389/fimmu.2020.564887/full). We found that HT-29 cells form spheroids with lower weight, diameter and size compared to other CRC cell lines. In the case that you are interested in increasing the sphericity and compactness of HT-29 spheroids I'd suggest using micro patterned ULA plates (Elplasia, AggreWell, SP5D) or adding 1:1 fibroblasts to your 3D cell culture.
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Are there any recommendations on how to safely remove the chamber from a chamber slide used for 3D cell culture (using reconstituted basement membrane matrices)? I am concerned primarily with shearing and/or distortion of the gel as the chamber is being dislodged, any tips/suggestions will be highly appreciated, thanks!
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I have used the chamber slides from Nunc.
You can remove the side walls easily with the applicator that comes with the box. Just keep in mind. You must be very careful and gentle while inserting the tool and lifting the plastic chamber. I always tried to have the gels in the middle of the well so that they won't attach to the sidewalls and then I have an easy process.
In the end, it is a matter of training.
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Hello everyone! I'm trying to grow porcine intestinal organoids from small intestinal crypts. I used 4-5 wks piglet for crypts isolation and organoids cultivation succeeds. However, when organoids were subcultured, some of them formed in different shapes compared with the primary organoid. I attached organoid images (Up: Images of primary organoids Day 1 to Day 5 / Down: Images of subcultured organoid on Day 4). It was similar to primary cultured organoid when many were cultured, but they spheroid shapes when few were cultured. I used organoid culture media (1xN2 supplement, 1xB27 supplement, 1mM N-acetylcysteine, 50ng/ml EGF, 100ng/ml Noggin, 2mM glutamax, 10mM HEPES, 100ug/ml Primocin, 10mM nicotinamide, 10uM Y-27632, 10uM SB202190, 500nM A 83-01, 2.5uM CHIR99021). In our lab condition, we used conditioned media (10% R-spondin media and 70% Wnt3A media) and Y-27632 inhibitor used first 2 Day. Give me any suggestion plz.
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3D Cell Culture is newly optimized and promising discovery in cell organization research and other researches that needs evindences in cell culture (in vitro) level. Are 3D Cell Cultures mimic epithelial cells because they are lining up the specific surfaces ? Is it possible to create an cell culture environment as we want in 3D cell cultures ? (e.g. microenvironment that show inflammatory responses as a result of cancer-related inflammation). Is real-time screening of cell response (e.g. after drug exposure) available in 3D cell cultures ?
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Thank you for your contributions to my question. It has developed my point of view. (like always) I wish good look to you too, it was nice to be your colleague :)
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Dear all,
I culture GSCs derived from patients. Few weeks ago I started having problem with them. They dont want to grow and make spheres. They are single cells
I culture them in Neurobasal A medium with 2% B27, 10ng/ml FGF, 10ng/ml EGF and 1.5 ml of GlutaMax.
I change only the medium from Neurobasal Plus for Neurobasal A medium.
Maybe someone have got similar problem?
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Hi all,
I am trying to wrap my head around splitting cells into different surface-area flasks. I could really use your help! I currently have 8 T-25 flasks of HEK-293T cells. I am familiar with splitting from a T-25 to T-25 or a T-75 to T-75. However, at some point, I would like to split perhaps 4 of the T-25 cells into T-75 flasks. Is that possible? If yes, how would the dilution be? A detailed explanation would be really helpful! Thank you in advance:)
Best Regards,
Mathangi
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Thank you so much Sidrah Chaudary and Malcolm Nobre ! This information is useful!
May I clarify if this would be the right procedure?
1. After washing, trypsinizing and neutralising the cells in the T25, I re-suspend them in 10mL of media.
2. Thereafter, I take 2mL of the suspension and add to 10mL media in the T75 flask. (for a 1:5 dilution)
Supposing I do not want to use too much media for the resuspension, can I just resuspend the cells in, say, 5mL media and add 2mL of this suspension into 10mL?
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Hello everyone,
I see a lot of papers with the spheroid generation topic. I know spheroids are used widely for simulation of tumor condition in body. Can someone say which abilities does the primary cells have which cell line do not have? like generation vascular endotheilal or producing extracellular matrix and which abilities cell line have which primary cells don not have?
Thanks in advance
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Hi all,
I have kept cells in -80 for several months, can I transfer them to liquid nitrogen now after all this time? I am afraid they would not survive.. I appreciate your input
Thanks!
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It should be ok. You can keep & use later on. - 80 degree C, commonly use for short time use e.g 1-3 months. LN for longer periods of time. Best of luck!
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I want to produce milk in vitro on a large scale. I read that oxytocin-mediated contraction of myoepithelial cells is necessary for the final expulsion of the MFG from the apical surface of the luminal cell. Is this always the case ? Are there other mechanisms for MFG expulsion? If there aren't other mechanisms, it means that I'll have to culture mammary organoids. This is not ideal because even if milk is, in fact, secreted, it will be encapsulated in the alveoli and hence hard to isolate.
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Dear Lucas House
Further Studies may be required to provide a scientific based answer to the question
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I am trying to analyse the size of spheroids and I am wondering which program is the most effective? I have found a protocol for spheroid sizer which is based on Matlab. But is there any way to analyze it also with the help of Image J or other programs?
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Hi,
you might want to evaluate a new image-based approach to compute multicellular spheroid volume available @
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Hello everyone,
I am using hanging drop method to make 3D cell culture. But I am struggling with handle the spheres when I exchange the media.
Please help me if you have any experience with this problem. Thank you so much!!!
Best,
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Do the aggregated cells stay in the floating culture afterwards? or they should be separated to prevent growing altogether? Thanks!
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I'm trying to grow PDX-derived cells from a SCCOHT model and I need them to survive short-term (7-14days) ex vivo
After tumor digestion I platted 7,000,000 cells in a 10cm ULA dish
In the following day they were forming spheroids but the spheroids were aggregating/clumping (First picture)
I filtered through a 40uM strainer and collect the portion that remained in the filter, washed with PBS, added 0.5mL of trypsin for 40s, neutralized with 1mL of 10% FBS media and them diluted in the serum reduced media (Counted with trypan blue and had >90% viability - Second picture)
I repeated this one more time after 3 days but they keep forming these giant aggregates every 2-3 days
I'm unsure if it is worse to separate them into single cells and lose the cell-cell contact or to let them grow in aggregates of spheroids
Does anyone know how to procced in this situation?
I digested the PDX tissue in Dispase/DNAse for 30min, filtered through a 100uM strainer, lysed the RBCs, minimized the debris with Ficoll and them platted in Advanced DMEM + 5% FBS
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Do you want to generate independent spheroids .
Varying concentration of serum with methyl cellulose
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Hi all.
I am totally new to this type of experiment- 3D culture. I am planning to study MCF10A cell line in a 3D environment. I am following these two publications as the protocol- doi:10.1016/S1046-2023(03)00032-X and DOI:10.1038/NMETH1015. I have some queries related to the method-
1) Are matrigel and EHS tumor basement membrane same in components? Because I found that, Matrigel is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS).
2) Is matrigel enough for acini formation with MCF10A cell line? Or, we need collagen in addition?
3) If I use cell culture dishes or multiplates for acini formation, how can I prepare the acini for immunostaining? Do we need to detach them form plate as ultimately we have to have a slide for confocal microscopy?
Thanks in advance. Your suggestions will be very much helpful for me.
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Thanks Mr. Javadi. I have started it on regular cell culture 60 mm dish.
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Dear experts,
I would like to work on THP-1 as adherent cells without changing its natural. Is there any method or protocol for develop adherence cell from suspension cell lines?
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Hello Dr.Stalin! In our laboratory we promote their adhesion by addition of phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng/ml. This conducts the differentation of THP-1 monocytes into macrophages. However, when you say "without changing its natural I do not know if you mean to preserve their monocyte phenotype". THP-1 cells are non-adherent, so all treatments you perform to promote their attachment are going to have an impact on them. For instance, PMA tends to upregulate the expression of some genes in differentiated macrophages, which could affect the gene expression induced by other stimuli. If I am not wrong and I remember well, the treatment enhances inflammatory genes through NFKB pathway. In our case, as we are studying inflammation, we added a period of arrest, leaving the cells without PMA in order to reduce that possible enhanced expression.
I would recommend you (depending on the experiments you have to perform) to try different concentrations of PMA in order to add the minimum necessary to promote their adhesion without enhancing the gene expression. Firstly, I would check the expression of inflammatory genes after the treatment and after the arrest, to see how the treatment affected your cells and whether the arrest decreased the inflammatory response. Lastly, I would characterize the cells through flow cytometry or gene expression of monocyte/macrophage markers to see the profile of your cells.
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Buddy Ratner @ University of Washington, US
Sheila MacNeil @ University of Sheffield, UK
Mitsuru Akashi @ Osaka University, Japan
Graca Raposo @ Institut Curie, PSL Research University, UMR144, CNRS, F-75248 Paris, France
Vitor M. Correlo @ Institute of Excellence on Tissue Engineering and Regenerative Medicine, Portugal
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Skin Tissue Models is giving a quite nice overview about currently published and/or commercialised skin tissue respecting different levels of complexity.
ISBN-13: 978-0128105450
ISBN-10: 0128105453
Published by
Alexandra P. Marques
Rui L. Reis
Rogério P. Pirraco
Mariana Cerqueira
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I'm currently using high concentration rat tail collagen 1 from Corning (product 354249). It is available up to 8-11 mg/ml max. I'm looking to get a similar product, but at higher concentration; preferably over 20mg/ml. Is anyone aware of such a product? The application is 3D cell culture in collagen 1 gels
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Could you make your own from powdered collagen? Sigma sell powdered type I collagen (#C7661).
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I need to transfer cell culture spheroids from agarose medium of a 24 - well plate to a histogel mold, to process them as paraffin embedded sections with IHC later on. The problem is, when I try to collect them out of the wells, they end up breaking apart. I hear about pipete them by cutting the tip of a S1000, or by covering the spheroids with a coat of gelatin and then extract them by cutting the medium, and even centrifugate the plate upside down to retreive the esferoids in the plate lid. How can do this, does it work? Is there any detailed protocol that can I use?
Beforehand thank you very much.
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Take a look at my publication where I specifically design a tool to perform 3D culture histology. Search the internet for 3D Cryo well insert. Its a well insert that can be cryosectionned.
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I am working on generating 3D construct using L929 cells. I have encapsulated the cells in a hydrogel matrix. After 4 days the cells have increased in number, however, their morphology is different. All the cells are round in shape and do not show elongated morphology of a L929 cells.
Does this happen because of the gel concentration??
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Aritra Chatterjee . the gel is very stiff, but gradually softens after 3 days. Is there any method to calculate the stiffness of the gel after prinitng.
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I will be infecting HeLa cells with C. burnetti. The MOI is supposed to be 50. Unsure how to go about calculating how much of the pathogen to add.
I have 1x10^6 Hela cells per well (in 3 mL of media) and the bacterial stock is 1.81x10^6 GE/mL.
I'm not familiar with using "GE" and not sure how to calculate. Thanks!
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Hey Casey,
the formula is always the same:
Concentration of your bacterial stock (cells/ml) divided by
Number of cells in well x MOI x Dilutionfactor of your infection volume
I would recommend to remove the 3 ml medium and infect in 1 ml or less depending on well size. We infect with 1 ml for 6- and 12-wells, 0,3 ml for 24-wells and 50 µl for 96-wells.
Lets say you infect with 1 ml per well. So in your case it would look like this:
1,18x106 bacterial cells/ml divided by
1x106 cells x MOI 50 x 1 ml infection volume
This gives you a dilution factor of 0,0236 for your infection solution, which is not very nice to pipette.
You can solve this very easily by increasing your bacterial stock solution concentration.
We dilute our bacterial stocks always to 1x109 cells/ml.
An example from our lab would look like this:
1x109 Listeria/ml divided by
500.000 macrophages x MOI 5 x 1 ml infection volume (in a 12-well)
The dilution factor for your infection solution would be 1:400.
Lets say we want to infect 4 wells, then you need at least 4 ml total. We take 5 ml as spare volume and then you get 5 ml divided by 400 = 12,5 µl bacterial stock in 4987,5 µl infection medium. Then you deliver approximately 5 Bacteria to each macrophage in the well in a volume of 1 ml.
In your case, I would not dilute your C.brunetti culture so much.
I hope this was not explained to complicated. Please ask for further explanation.
All the best,
Marc
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Can anyone recommend me a method or software to quantify the average amount of PI staining in tumorsphere relative to the size of the tumorsphere by using confocal images?
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Hi,
I assume that you have taken stacked confocal images of the 3D tumorsphere and want to count the number of PI stained cells?
It can be done via ImageJ. These are some methods mentioned in papers. This is one that use ImageJ to quantify viability through staining and confocal images for 3D culture. They also have a macro for that: Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M., Sprecher, C. M., & Ito, K. (2008). Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds. Tissue Engineering Part C: Methods, 14(4), 353-358.
Or you may search along with those keywords if you find the method makes sense but it is not ideal for you.
Alternatively, if IMARIS is available to you, it can be used to analyze 3D images directly. Basically you need to set an appropriate intensity threshold and generate particle volumes. Similar to ImageJ, there will be a whole list of particles generated (but it is 3D here), with information of sizes, intensities etc. Then just record the number of particles (that is the number of PI+ cells). Often, you will find 2 or more particles stuck together, then you may use different methods such as watershed to separate them. Overall, it is similar to 2D image analysis, and if you use a macro or program to count them, there will be some errors, but if you set the parameters/thresholds right and use them consistently, you should be able to get a fairly close number.
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Hi experts,
I wonder if there is a possibility to seal well plates for cell culture in a sterile environment in way that liquids can remain in the well plates? Something like vacuum packaging where the lid is packed separately?
We have a hydrogel which should not dry out and ideally is immersed in buffer all the time. Now, we cannot transport the well plates far, since there's leaking of the liquid from the plates if tilted.
Sterility must be maintained.
Thank you very much!
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Hi Marcus,
my first guess would also have been parafilm + the lid of the dish.
However, one question would be what size your plates have and how far you need to transport/how long you need to store them. Sealing small wells with 2 layers of parafilm, fixing the parafilm afterwards with the lid and storing in the fridge worked well for me.
I used to use parafilm as sealing a lot for flow chambers and had never any issues with sterility (sprayed it with EtOH and dried in the flow cabinet).
Katrin
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Hi All,
I was wondering if anyone had experience of working with automated systems for drug dosing for discovery experiments. I want to run ADME-tox type experiments on multiple drug combinations, ideally in a medium-high throughput format, but I don't want to do it by hand and want to automate the process of medium change and drug dispensing at different doses/combinations simultaneously.
Does anyone have experience doing thins kind of work, and if so with what equipment?
thank you
Fabio
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iam sorry, i dont have experiance.
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I am performing tests by adding increasing concentrations of drugs to my cell culture media (using DMSO as a vehicle), but in the higher concentrations especially I see these large dark cloud appearing around my aggregates. It seems to correlate with increasing drug concentrations, so I thought it might be to do with a stress response or cell death but can't find any literature to help. I don't think it's infection as it doesn't resemble any typical infections, and it doesn't look like the drug precipitate as I've seen that before (this one I'm not 100% sure about). It seems to closely correlate with the toxicity results I get from the LDH assay too. Cell line is HepG2 Any ideas?
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It is difficult to tell from the image without seeing it in relation to the other dosed samples. However, it resembles what we classify as a type of flocculent precipitation that is distinct from the crystalline precipitation that is more common at high doses. 150 mM is a fairly high working concentration for any compound, so I would expect to see some precipitate at or near that dose level in many test compounds. If you are seeing increases in LDH without any significant reduction in cell viability than the precipitation could be interfering with your luminescence readout.
Best,
Nick
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Hi everybody,
I have made cryosectioning from 25% methyl cellulose harvested spheroids and when they were one month old, I prepared cryosectioning slides of them, the slides shoulkd show a round shape composed of monolaer cells a round and big empty hole inside , but my sample showed the center of spheroid filled of cells inside. Does anybody know what is the problem?
thanks for your help
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Have you cut spheroids at different timings, i.e., 10 days, 20 days, May be be hole appears initially and gets filled at later timings. What is the wrong with tif the hole gets filled, just curious.
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Hi everyone,
I wanna use Cell titer Glo for my spheroids, my spheroids are in the diameter between 350- 390 micrometer, in the protocol it is written the 100 micro liter is enough for every spheroid, but I have heard it is possible to use less amount of it, if someone has information and experience using it previously I would appreciate it if tell me how much is enough for this size of spheroid?
also if it is needed using negative control and positive control, I would appreciate explaining how should i prepare them?
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Hey Elmira! Try sending it to de_techserv@promega.com. The techserv in Switzerland, typically replies within 24h or less. If the German one doesn't answer you, let me know and I'll ask the ones in Zurich.
I am not sure about my answer and so I think it's best to check with them. I don't want to have you waste reagents and time.
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Do I need to convert 3D tumorsphere into single cell suspension before the alamarBlue treatment in assay? Or else can I use the intact tumorsphere directly for the alamarBlue treatment? Are there any related published protocols?
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Hey Janith,
Why are you focusing on AlamarBlue as a viability assay? Are you open to using others? Promega has many products dedicated to 3D cell cultures. If I were you, I would have a look there and see if anything seems good. They even have samples for some of their products so you could test some for free too :
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We are working with Ovarian cancer cell lines. we want to find the viability of our cell lines (in present of our nanoparticle) in 3D. Also, I need to do 3D live/fixed cells imaging.
I found a lot of protocols( Alvetex, RAFT 3D culture...) but it is not still clear to me. should i dissociate the cells from scaffold and do the reading? Can you suggest me a good and reliable protocol that you got result from that? Thank you.
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Zonghan Gan Can you give me the protocol that you are using for your experiment? Thank you
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Im co-culturing the caco2, Ht29-MTX cell lines (ratio 1:10) on a silicon porous membrane by the pore sizes of 6um (center-center 50um). Actually I dont use the SERUM-FREE media and I do not know why do they use it in litreture for caco2 cell lines cultivate.
The result is that I see the cells in the holes but they are almost stoped to pass to the other side. And on the top surface of membrane, there are only clusters of cells instead of the cell monolayer.
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Try using Serum-Free medium.......
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Hi,
As harvesting spheroid from primary cells takes longer than cell line and as hanging drop method is best method among others is best for spheroid formation, the changing medium would be an serious issue. if someone used hanging drop for producing spheroids, can someone explain me how and after how many days the medium was changed for their spheroids?
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Do we still use such methods? It is too old method. Nowadays, you just have to use ultra low attachment flasks or nunclon sphera flaks and you can easily get sphere.
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Hi everyone,
I have tried to generate spheroids from primary normal RPEcells using methylcellulose in 96 well plates _U bottom based on some papers but it doesnot work.
if some one has experiences with methyl xcellulosec an tell me how much concentration did they, to get good result?
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First you seed the cells in a dish without coated with attachment factors. after 5 days the round shaped sphereoids appered. collect those. let them settle under gravity. then plate 5-7 spheroids on inducing environment for differentiation.
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I had problem with crumpled cells after tissue digestion.
After adding 2 ml Adv DMEM/F12 to the minced tissue, I used 2 ml accumax and 100 ul collagenase XI (20 mg/ml) together to further digest the patient's cancer tissue for about 1.5 hour (in 37°C incubator) while pipetting every 15-20 minutes.
It's dissociated well but I got very sticky cells crumpled together, even after I used cell strainer as well. It was so difficult to pipette. And it became worse when I added the gel and wanted to drop it to the plate.
Please help me, does anyone have this experience before?
Is it because I used too much of accumax/collagenase? Or too long digestion time? I wonder whether I can use DNase? How much should I add DNase?
Thank you
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Dear Ariestya,
Yes, it sounds like you are liberating a lot of DNA using your dissociation procedure.
It is very common to use DNase I in the digestion of brain tissue in order to get single cells for primany neuronal cell culture. For neuronal cell work, one must also add a DNase inhibitor at a certain point. I have found that how much sticky DNA is released will depend on the tissue and the method used for physically dissociation. For neurons, I am very gentle, triturate with Pasteur pipettes with sequentially smaller openings, and usually have some sort of carrier protein like BSA in the dissociation media to help protect the cells and help keep them from clumping. I also avoid trituration of cells that have already been dissociated. So I triturate until the media becomes a bit cloudy, let the large chunks settle, and then pull of the suprnatant with the single cells and keep that in a separate tube. I repeat this procedure two more times with successively smaller bore pipettes, combining the supernatants. I find that for viable neurons I should quit at that point, since any neurons I recover from the remaining tissue are usually dead by Trypan Blue exclusion.
Since tumors are different from brain tissue, you might want to check a few dissociation protocols in your field for suggested times of enzyme exposure.
Good Luck!
Jill
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Hi all,
I am coating my plates with poly-HEMA for spheroid culture and I have a couple of questions for those of you experienced with this type of coating. Firstly, when you are drying the plates overnight in an incubator - do you use your normal cell incubator with cells still in it? Secondly, does this protocol sound appropriate? I had some smudging but perhaps it is because when I first did it I filtered the solution following step 3.
  1. Pre-heat ethanol to 65°C
  2. Dissolve 1g of Poly-HEMA in 50ml ethanol pre-warmed to 65°C (Add EthOH first and then add the powder!)
  3. To completely dissolve. Leave in 65°C water bath overnight (agitation helps to dissolve the Poly-HEMA e.g. shaking water bath)
  4. Add 200ul of 20mg/ml Poly-HEMA to well of a pre-heated 24-well plate (tilt plate to homogeneously cover area) and place in incubator overnight with lid on. Wash wells with PBS x3
  5. Seed cells at desired concentration
Thanks!
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Hi Laura,
Here is a protocol used routinely many years ago in the lab:
A 12% solution of polyhydroxyethylmethacrylate polymer (poly-HEMA) in 95% ethanol was mixed overnight, centrifuged at 2,500 rpm to remove undissolved particles, and diluted 1:10 with 95% ethanol. 100-mm dishes were coated with 4 ml of poly-HEMA solution and left to dry at room temperature (inside the biosafety cabinet under sterile conditions with the lid half way on. Dishes were washed twice with PBS and once with HBSS before use. Cells were typsinized, resuspended, and added to the polyHEMA coated dishes at a desired density in serum containing medium.
I hope it helps!
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Hi, I am creating a 3D model for in-vitro drug screening. In one step, I want to create a hydrophobic surface on the coverslip, which is needed to have resistance to acetone and also need to biocompatible for culturing a different kind of cells. Can anyone suggest me a product to create such coating?
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Dear Arunkumar Rengaraj,
You may use PVA coating for your work. It did successfully coat in plastic based plate but for the glass slip, you should try first. Good luck. Thanks
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I would like to know the viscosity of Matrigel (Corning) as function of temperature. I already know that it solidifies at 37° and its liquid at 4°, but i would like to know what's the transition form, and what happens with different Matrigel concentrations.
Thanks in advance,
Simon
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I had put some of the Matrigel I was culturing my HUVECs on on a rheometer a while back, and it had produced a pretty flat G'/G'' from 20ºC to ~40ºC. I don't exactly remember the details, unfortunately.
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I recently have imaged a 3D-cell culture of HEK-293T cells stained with DAPI in confocal microscope. Cells are readily visible in 10x and I find difficulty in finding speroids in 63x oil, also noise is a major issue. Suggest me something for a better imaging of a 3D- culture.
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you can also embedding your cells in fibrin or Martigel.
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Hi, I have grown adherent cells on glass coverslip and would like to use them for Immuno Fluorescence experiment later. I would like to know if it is possible to dehydrate the cells gradually using different percentage of ethanol and store them for now, which I can fix later and use. I heard that fixing the cells and then storing them might lead to detachment of the cells if the fixed slides are more than a week old. Can someone help with the protocol, if any please?
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hi, during your protocol, did you carry out dehydration with increasing concentration of cold ethanol following FA treatment?
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Hi
I have a PHPMA hydrogel which is synthesis from HPMA + MBA +AIBN in aceton/DMSO. It swells in water and PBS and DMEM perfectly in room temperature and in refrigerator. But when I put it in a 96 plate and incubator it start to deswell rapidly and shrink.
What can make this hydrogel thermoresponsive like this?
Based on papers, it should not show such behavior
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I found this interesting device (https://darwin-microfluidics.com/collections/microfluidic-organ-on-a-chip/products/ddi-chip-distance-dependent-interaction-chip-pack-of-3). Have you any experience in the use of microfluidics to study cell cross-talking and signaling?
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I did some research on 3d cells migration through microfluidics but not know much about cell cross-talking. This article would probably be helpful to you https://www.nature.com/articles/s41598-018-30683-4
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Hi everyone,
I am working in the role of Wnt signal and spindle orientation.
I am working with mouse organoids from colon but I would like to have a simpler system to test some idea. In the lab we use CaCO2 cells that form 3d cyst but they are human cells.
I was wondering if someone have experience with mouse cells that can create cysts such as in CaCO2 cells.
Thanks
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It is a created environment in which l cells are interact with their surroundings in all three dimensions. Thus 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These cultures are usually grown in 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.
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I am trying to improve the survivability of primary neuron cell cultures. Currently, I am getting great survival out to 20-days but would like to push my time point out to 42 days. I currently believe that the neurons are being crowded out by microglia so I am looking for ways to reduce their growth. Additionally I am curious if anyone has had any experience with 3D cell culture techniques and could provide some technical expertise and thoughts on this method compared to standard 2D cell culturing.
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1. The way to prevent overgrowth of glial cells is to remove FBS. You may grow the astrocytes with DMEM + FBS, then you have to use the same volume of Neural Basal A plus B27 for seeding the neuroprogenitors. 2 to 3 days later, you may have to have all the medium without FBS.
2. Someone said 9% of oxygen may help to survival of cultured neurons.
3. Depend on what types of neurons you are growing. If you were starting with cortical or hippocampal progenitors younger then E12.5 of mice, I would suggest you to add certain amount of GABAergic interneuron progenitors from MGE and CGE to make a "natural" population environment.
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I am trying to develop 3D cell cultures with fibroblast. I am hoping to use a collagen method described by Bott et al., 2010. I just want to know, when we prepare the collagen gen and insert the fibroblasts, if we need to confirm that what we prepared was actually a 3D model of cell culture. And if there is a method to do that. Thank you
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you can use optical sectioning to see your cells in your 3D gel. A nice confocal microscopy will do the job.
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Our lab has fabricated graphene based 3D scaffold for cancer cell growth. We have checked for viability of NCC Rbc51 and MCF7 cells for up to an week. We want to study the gene expression changes between the culture plate and 3D grown cells. We expect to know the relative closeness of the scaffold grown cells towards an in-vivo tumor/xenograft/organoid. Please suggest me the time point or experiments that I can perform to choose an ideal time point to fulfill the goal of the study.
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Hi dear Dr. Kumar,
The time point is important, however other factors are still important such as the confleuncies in 3D vs 2D to create standarizations.
Concerning the time point, 24 hours is enough to analyze the cells in 3D model, simply just allow the cells to interact with the external cellular matrix in 3D model.
Feel free to contact me if you have any more qeustion, please
Best wishes
Sarmad
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Dear colleagues,
I wanted to start a discussion regarding the advantages of 3D cell cultures. If you are currently using 3D cell cultures, what benefits do you believe they have to your research? Additionally, in what ways do you think they can be improved?
If you are not currently using 3D cell cultures, is there a reason you are not?
I am currently working with different cell culture techniques and wanted to understand the community’s opinions on the different systems! Please contact shailyshah@gatech.edu for further comments on the topic! Your thoughts are greatly appreciated.
Shaily Shah
Biomedical Engineering Intern
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Ahmed A. Askar In your reply you give an overview of only spheroid aggregates, which is a valid 3D cell culture technique, but there are several other more complex techniques that are capable of producing more tissue-like cultures including:
  • Organ buds;
  • Re-cellularised organ matrices;
  • Tissue patches (for heart and skin repair/research;
  • Micro-tissues created via biofabrication (such as bioprinting).
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I want to know how to prepare sample for HPLC from 3D-cell culture for cancer drug analysis.
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Dear Varun Ahuja
Thank you for your answers. I will read the attached papers.
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Previously, I used to seed primary dermal fibroblasts (which initially had slow growth rate) in collagen hydrogel and the cells used to show complete attachment and the normal shape of fibroblasts after culturing the gel in complete media. Now I have new fibroblasts which grow fast in flasks and once seeded in the hydrogel, they show some attachment within few hours but then start to take the circular shape and make colonies which make any further tests difficult.
I have tried different collagen concentrations (1, 1.5 and 2 mg/ml) as well as different cell densities (5000, 10000, 20000, up to 100000 cells per 50 and 100 microlitre hydrogels, but the behaviour of the cells were the same.
Please, could you explain how I can overcome that problem?
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Dear Amir,
I have worked with these hydrogels but used stem cells. If you are putting cells inside the gel, they will die very fast and don't really proliferate inside the gel. I compared both methods.
The better way is to coat gel first on plate wells and then seed cells on it's top. They will still grow in 3D conditions. Cells really grow well in those conditions.
Hope it helps!
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I am using the TeSR-E8 medium for the maintenance and growth of iPSCs. I made the aliquotes of complete medium without adding Pen/Strp and Normocine, and stored at -20C. On need, I thaw one of them and store the remaining volume at 4C where it remains free of contamination for one week. But it gets contaminated after that even though I use and store the vial very carefully. Can I please get any suggestions regarding the use and store of stem cell medium? Is it ok for iPSCs to use medium containing Pen/Str and Normocine?
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Hello
We routinely culture iPSC in medium without any antibiotics but If you need to use them, it is recommended to use lower concentrations ( 1% pen/strep )
Good Luck
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Dear All,
Does someone know how we can shrink the number of days in vitro for neuronal differentiation protocols? According to the literature, it takes 40+ days (starting from human ESC/iPSC) to get more or less differentiated neurons.
Most of the protocols are employing different growth factor such as BDNF, GDNF and NT3 for neuronal maturation but it still takes plenty of time.
Any help appreciated.
Thank you in advance!
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Hi,
I am sorry to tell you that it takes more than 40 days when you use human cells. Its depend on which stage of differentiation you start (ESC, hiPSC; NSC, early neural progenitors (eNP), neural progenitors (NP)).
When you start from ESC, hiPSC at the undifferentiated stage you have to obtain:
1. Neural roset(NE) from hiPSC, ESC (~ 15-21 days);
2. Select neural rosettes(NE) and prepared expansion of neural stem cells (NSC) (~ 6 weeks) to get the stable NSC phenotype;
3. Early neural progenitors(eNP) from NSC (~21 days);
4. Neural progenitors (NP) from eNP (~14 days);
5. Mature neurons from (NP) (~90days);
I use this protocol for hiPSC neural differentiation. It takes a long time, but at the stage of NSC, you can freeze the cells. Freezing in later stages of differentation (eNP, NP) is not recommended.
Good luck!
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What do you think about progress in 3D cell culture systems?
Please find our short questionare about 3D cell culture trends.
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Sure, the results will be shared here
:-)
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I am trying to grow SH-SY5Y neuroblastoma cells. or main objective is to get the dendrite and axon structure and neuronal network like growth. We are getting the small connections between neuron to neuron,but it takes 10 days to get even minor connection. In the complete plate we are getting in some places such growth, where as maximum time we get clump growth. So can any one suggest any technique to get neuronal network based growth?
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See, for instance, a lit of medium in H. XICOY et al.,Molecular Neurodegeneration, 2017, DOI 10.1186/s13024-017-0149-0, and especially the reference 11, 19 et 29 which give relevant details for obtaining neuronal différenciation;
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I'm preparing scaffold to perform 3D cell culture. I'm following up the Silk fibroin protocol and I'm stuck here with a technical issue. I have added 3g of silk in 12mL of 9.3M LiBr solution and I'm maintaining 60'C as mentioned in protocol. Even after 4h there's literally no sign of solubility of silk in LiBr solution.
Kiindly help me to troubleshoot this issue.!
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Dear Kishore,
I recommend to recheck the concentration of your LiBr solution. It really has to be 9.3M. E.g. 80.8g sould be dissolved with water to 100mL total volume. If you add 100ml to 80.8g of LiBr you will not get a 9.3M LiBr solution. And then the dissolving does not work efficiently or at all.
Another possible problem might be the degumming time. How long did you degum and with which reagent? If you are using Na2CO3 then the longer you degum the faster the dissolving process in LiBr is.
Best regards,
Andi
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I want to know the reason why the number of cells are different to test the same drug.
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These two cell lines (MCF-7 and MDA-MB-231) have different biological features and behavior (they represent differemnt types of human breast cancer, express differenmt markers, possess diffrenet growth and invasive characteristics). And we need to optimize the 3D cell culture conditions of each individual cell line .
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I've done hanging drop with A549 cells. (10 ul/20.000 cell/drop) At second day I saw a few cells have attacht on lid, behind the sphere colony. At third day they become much more cells that attachtes on lid. Does anyone have ever experienced like that? How can they attacht on lid? 1) I don't think there is no Poly-D-Lysine on lid and 2) How did they withstand gravity?
I'd expect the cells would attacht each other and going down because of gravity but some of them going up and attacht on lid.
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Thank you for your advice Harnath Shelat. I will try it as soon as possible.
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So I am doing an experiment involving endothelial cells (HMVECs) plated on top of Matrigel and am trying to look at the branching and morphological properties of the cells over a time period. I have been using Matrigel and see tube formation and structures very rapidly and want to slow down this process. Are there any known protocols for this such as a mixture of collagen and Matrigel? If so can someone help me find references.
I'm looking to see slower tube formation over a course of days not hours.
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Hi Danielle, have you seen the real vessel tube like structure after ECs culture on matrigel? How long did you culture? My guess is that ECs might group each other resembling vessels, what do you think? I may be wrong, ECs sprouts might form vessel like structure, in fact it might not be a real vessel. If there is a real vessel formation, you can control angiogenesis either by reducing the ECs numbers or using low concentration of matrigel or reducing both ECs number and matrigel concentration. Please update your successful attempt on how you control the rapid angiogenesis on matrigel substrata. Thanks.
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I am studying vasculogenic potential of cancer stem cells, and so I culture stem cell spheres on Matrigel. After several days, they form 3D tube structures into the gel.
Inhibition of my protein of interest prevents this tube formation, so I want to interrogate the various angiogenic kinase pathways to see what is being changed between my control and my treated group.
However, phospho-RTK are notoriously unstable and so traditionally Western Blot cell collection has to be done very quickly on ice to preserve the phosphorylation.
What I need is a method to get my cells out of the matrigel (its a soft substrate and they grow INTO it, so I can't just scrape them) so I can lyse them without actually changing them. Accutase (or Trypsin or Versene) won't be effective because a) they can degrade the extracellular RTKs and change their activation and b) it takes too long at 37oC so receptors can lose their phospo groups.
BD has a 4oC cell recovery solution but it takes an hour - could this potentially work?
Thank you for any help you all can provide!
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Just telling you what we do in our Lab.
Matrigel gellify at 37°C but this process is reversible simply return your plate, with your tube-like structures, to 4°C o.n. The day after applying pre-refrigerated PBS to dilute Matrigel and cells at least 20X and transfer in tubes. Then collect your cells by centrifugation at 400g 5'. After a further wash in PBS, the pellet could be processed for protein extraction.
Hoep this could help somehow
Good luck