3D Cell Culture - Science method

I always use Aprotinin to keep the cells from degrading the fibrin

You can perform the WST-1 test directly on alginate-based 3D colorectal cancer cell cultures without removing the alginate spheres. The WST-1 reagent can penetrate the 3D structure, allowing the assay to measure cell viability within the 3D environment. Here are some tips:

1. **Ensure Adequate Reagent Penetration**: Mix the WST-1 reagent thoroughly with the medium to ensure it can diffuse into the alginate matrix.

2. **Incubation Time**: Allow sufficient incubation time for the reagent to react with cells within the 3D structure.

3. **Readout**: Measure absorbance as per standard WST-1 protocols.

This approach allows you to maintain the 3D culture conditions and assess cell viability more accurately in the 3D context.

It is important to also consider that the availability of oxygen to the outer layer of cells is different to what the inner cells in the 3D culture are receiving.

Poor growth of mouse liver organoid after each passage could be attributed to a variety of factors. Organoid culture can be sensitive and complex, and any changes in growth patterns should be carefully investigated. Here are some potential reasons for the observed poor growth:

1. Genetic Drift: over time and passages, genetic mutations or changes may accumulate in the organoids, affecting their growth and function. This can lead to reduced viability and growth rate. It’s important to periodically verify the genetic integrity of your organoids using techniques such as genetic typing and DNA sequencing.

2. Contamination: contamination with bacteria, fungi, or other microorganisms can significantly impact the growth of the organoids. Make sure your culture equipments and reagents are properly sterilized and periodically tested for contaminants. Contamination can also occur during passaging, so maintaining aseptic techniques is crucial.

3. Cell Quality: the initial quality of cells used to establish the organoid culture can impact long term growth. Poor quality or low viability cells can lead to poor organoid growth overtime. Ensure that your starting cell population is healthy and viable.

4. Media Composition: the composition of the culture media can greatly influence organoid growth. Changes in media formulations, growth factors and supplements can affect the organoids’ ability to proliferate and differentiate. Double – check and be sure that the media is consistent in each passage and stock.

5. Passaging Techniques: improper passaging techniques can damage organoids, impacting their growth. Be gentle during dissociation and ensure that the enzymatic digestion time is optimized to avoid excessive cell stress.

6. Substrate and Matrix: the substrate or extracellular matrix used to grow the organoid can impact their growth and differentiation. Make sure you are using the suitable substrate that provides the necessary cues for proper organoid development.

7. pH and Osmolarity: fluctuations in pH or osmolarity of the culture media can affect the health of the organoids. Regular monitor and maintain the appropriate pH and osmolarity levels.

8. Freezing and Thawing: if you’re using a frozen stock of organoids, improper freezing and thawing techniques can damage cells and lead to poor growth upon revival. Use proper cryopreservation techniques and test the viability of the thawed cells before proceeding.

9. Adaptation to New Conditions: when establishing a new stock or transferring organoids to a different laboratory, they might need time to adapt to the new environment. Gradually acclimatize the organoids to any changes in the culture conditions.

10. Cell Lineage Instability: some organoid cultures can exhibit lineage instability, where cells start to deviate from their original tissue types. This can lead to changes in growth behavior and differentiation potentials.

To troubleshoot this issue, you should carefully review and document your entire culture protocol, including media preparation, passaging technique, and any changes that have been introduced. Systematically address and test each potential factor to identify the root cause of the poor growth. Collaboration with colleague or experts in the field can also provide valuable insights.

I agree with Phillip's comment on the hypoxia at the core of 3D cultures. This will be diffusion dependent, determined largely by the size of the organoids/spheroids and will be very difficult to measure in vitro.

I've not come across genuinely perfused 3D cultures yet, which would be another way to control oxygen concentration.

Perhaps an empirical approach could be taken by IF staining for oxidative stress markers for example.

Hi Genesis Marrero

Interesting observation! Based on your description, it seems like the crystals could indeed be salt precipitates. There could be several reasons for this, here are a few possible explanations. Media evaporation: If your cultures are not fully sealed or the incubator is not properly humidified, evaporation could cause the salts in the culture medium to become more concentrated over time. This might lead to precipitation, especially near cells which could act as nucleation points for crystal formation. Interaction with the disc material: Calcium phosphate could be reacting with components of the culture medium, leading to formation of insoluble salts. I hope these ideas help you in understanding and investigating this phenomenon further!

Hi Devin,

I hope that you are well!

Sorry for the late reply, I came across this question whilst looking for information on AC16 spheroids.

Yes this technique of producing spheroids with AC16, endothelial cells and fibroblasts is possible. It is a model I have used in my PhD and can produce some nice data.

If you are still interested in this technique, I would be happy to further discuss.

thanks,

Rob

I have not worked with this drug before, but being a small molecule, it should possess appropriate physicochemical properties to enhance its trafficking through the ECM and indeed, the organoid itself. But bear in mind that the drug has to overcome 2 barriers: the matrigel and the organoid. You make do a pilot trying different delivery systems e.g direct delivery via microinjection, nanobased delivery etc

Of course, time is a significant factor for spheroids' size and viability of cells, due to when you seed the cells to the generation of spheroids after the cells are in the proliferative stage. More time (days) can lead to the big size of spheroids, in addition, the cells located in the core zone are suffered from nutrients, oxygen, and ...

I suggest you set up the best time to achieve reliable results too

Probably you may need to check with the apparatus or materials your using for the preparation of 3D spheroids.

Check the materials/gel you're using for spheroids preparation by placing them in a sterile medium with FBS. Leave them for a few hours (24 hours - 48 hours). Afterwards, check in the microscope to identify contaminants.

It will be easy if you can give the details asked by Davide Confalonieri

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.

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!

If you want to grow spheroids, you can use 0.6% agarose-coated well for cell growth. Jarshad Jas

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.

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.

Dear researcher

See the following papers method

Cell viability analysis using trypan blue: manual and automated methods

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

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:

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:

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.

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

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

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!

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

I suspect the amino acids in the medium will compete for chelation of the calcium ions.

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.

you could try to use a low-melting agarose.

Simply, go where the most and best food is available. :-)

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

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.

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.

have you checked this?

Dear Begüm Kurt ,

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 :)

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?

I wish this link helps

https://sciencellonline.com/blog/primary-cells-versus-cell-lines/

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!

Dear Lucas House

Further Studies may be required to provide a scientific based answer to the question

Hi,

you might want to evaluate a new image-based approach to compute multicellular spheroid volume available @

Zhonggang Feng

Do the aggregated cells stay in the floating culture afterwards? or they should be separated to prevent growing altogether? Thanks!

Do you want to generate independent spheroids .

Varying concentration of serum with methyl cellulose

Thanks Mr. Javadi. I have started it on regular cell culture 60 mm dish.

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.

Could you make your own from powdered collagen? Sigma sell powdered type I collagen (#C7661).

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.

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.

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

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.

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

iam sorry, i dont have experiance.

2D models will be easy to establish. 3D models are difficult to establish but will give better results, as they will have more structures interacting with each other, and thus more closer to physiological system.

Hi Ben Allcock ,

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

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.

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.

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 :

Zonghan Gan Can you give me the protocol that you are using for your experiment? Thank you

Try using Serum-Free medium.......

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.

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.

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

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!

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

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.

Punita Bathla even I use Matrigel and I image them in 10x/20x and that worked well for me so far.

hi, during your protocol, did you carry out dehydration with increasing concentration of cold ethanol following FA treatment?

It is a known phenomenon. See Figure 1 in https://www.researchgate.net/file.PostFileLoader.html?id=5565600f6225ff4f6b8b45e0&assetKey=AS%3A273784404152346%401442286696885

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

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.

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.

you can use optical sectioning to see your cells in your 3D gel. A nice confocal microscopy will do the job.

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