Tumor Cell Culture - Science topic

Hi guys,

I'm working on the tumor mouse model. In general, I prepared tumor cells and injected cells into the flank of mice subcutaneously.

After around 3 weeks, I found, in the same group, there's a variation in the tumor weight. For instance, some tumor were around 1.2g, some 0.6g.

Could you give me some suggections or tips about how do tumor inejction?

Which type of needle do you need for injection? or How to prepare tumor cells suspension? or whatever something you think is important to do that successfully

Many thanks :)

Hello,

I am trying to transduce my CD4/CD8 T-cells with lentivector using retronectin from Takara (#T100B) at 50ug/ml, after they are activated by CD3/CD28 for 2 days.

The official protocol says that "it's important that the target cells be in logarithmic growth phase", but how this state may be monitored?

How to choose the "perfect moment" for transduction via retronectin?

Thanks in advance!

Need this cell line urgently....

Dear brain tumor and U-87MG gurus,

Greetings!

Recently I bought a vial of U-87MG from ATCC and started culturing them following ATCC's SOP. However, I ran into a problem that the cells tend to grow in big spheres (attached pictures 1, 2, 3), rather than the nicely spreadout monolayer shown in ATCC website (attached picture 4). This bugs me a lot as I'm not sure whether I have a problem here, especially after comparing the morphology of cells I got with the one posted on ATCC wesbite. I'm wondering if anyone had this issue and would much appreciate for any insight.

Details about the culture:

1. Media: I have used ATCC's recommended media, which is ATCC's EMEM+10%FBS (not heat-inactivated)+P/S. I have also used Sigma's high-glucose DMEM+10%FBS (not heat-inactivated)+P/S+GlutaMax+Na-Pyruvate+NEAA+HEPES. However, both conditions resulted in similar problem as mentioned above. 

2. Plates: I have used regular TC-treated plates from two different vendors (TPP and Sarstedt), and the issue happens in both.

3. Seeding density: 4x10^4 cells/cm2, i.e. seeding 6 million cells per 15cm plate

4. About attached pictures 1/2/3: seeded in 15cm plates as mentioned above in Sigma's high-glucose DMEM+10%FBS (not heat-inactivated)+P/S+GlutaMax+Na-Pyruvate+NEAA+HEPES for 5 days. Media change every other day didn't seem to address or improve the issue.

5. I sent the attached pictures 1/2/3 to ATCC tech support for consultation, they concluded I'm having contamination, as judged from the morphology of cells in the culture. I have not tested mycoplasma contamination for these cultures yet.

6. I've also tried longer trypin treatment time (20min at 37C), which seemed to help the cells to spread out nicely on the 2nd day, however, the problem came back on the 4th day.

7. The culture shown in the pictures is about the 10th passage of the cells from the received vial from ATCC.

8. I had some experience on adherent culture of glioma stem cells on laminin-coated plates. Those cells will appear like the pictures 1/2/3 here if I grow them in regular TC-treated plates. I guess I'm not surprised to see U-87MG is growing in a similar way with big spheres on TC-treated plates, but the ATCC culture picture destroyed my guess here with very nice monolayer culture....

Thank you very much in advance for your time, consideration and help!

Best,

Kai

Please take a look at the attached file.

I irradiated cells using a fractionation regime of 3 x 1 Gy after exposure to a substance in different concentrations.

I made an XY table with the determined SFs and plotted a graph using the LQ-model.

The equation I used was Y=exp(-3*(A*3*X + B*3*X^2)). Its an edition of the provided equation Y=exp(-1*(A*X + B*X^2)) in regard to the fractionation regime.

To determine the AUC I used the standard analyzing tool that Graphpad provided.

Could someone tell me, if this is right or if I mistaken somewhere?

Tank you very much in advance!

I have recently started cell seeding inside my PDMS microfluidic device with syringe pump. I am actually trying to form tumor spheroids inside microwells coated with pluronics. However, the MCF7 cells instead of forming spheroids are attaching to PDMS surface. Please let me know if you have faced similar problem while forming spheroids on PDMS microfluidic devices

I am processing high-grade serous ovarian cancer samples and trying to grow tumor cell culture for subsequent functional assays. Any recommendation on the dissociation process, enzyme mix and/or culture media?

We did qpcr using our sample tumor cDNA to compare with four different normal human tussie (colon, prostate, thymus, and spleen). I've calculated the 2^-ΔΔCt to be 7.08, 3.10, 0.17, 21.2. I understand that this means the gene expression level of the tumor sample is 7.08, 3.10, 0.17, 21.2 times of control tissue. I'm not sure if how much 2^-ΔΔCt would suggest mutations and can I conclude this to be tissue specific changes as the highest result is in spleen?

Dear All,

I am taking over the Master's student's project and I am a new PhD student. We have experiments involving tumour implantation in mice. The MSc student is used to scraping cells down when passaging and only uses trypsin to detach the tumour cells for implantation.

In my previous lab, I was taught to trypsinise as it is less damaging to cells when passaging and that apoptotic cells could influence culture environment. Therefore, I have been using trypsin (5 minutes, 37 degree C). However, I have noted a difference in cell growth as the MSc student is used to passaging at very, very low concentrations of 1:1000 whilst I resorted to passaging 1:10 every 3-4 days. I have been keeping track of the growth rate of my cells which is about 3.1-3.7% (quite consistent) but they seem to be growing much slower than the MSc student.

I consulted the MSc student and I was told to swap to scraping. I am just curious as to how this may affect the cells and whether anyone has ever noted such a huge difference.

Note: We re-use the T75 flask (hence, why I decided on trypsinising to cause less damage to my cells).

We mostly run FACs and IHCs in our lab if that will help determine which method would be more suited to our cause.

Thanks.

Hi, I am currently design a assay for testing the cytotoxicity of primary T cells against tumor cell lines. Here is the design:

Cryo-preserved PBMCs were retrieved and cultured with 9ug/mL PHA, 20%FBS RPMI1640 medium for 48 hours. Then the PBMCs were stained with CD3 antibody and were sorted for CD3+ T cells. The adherent tumor cell lines A549 were seeded the day before add T cells in 96 well plate (3000 cells/well). Tumor cells were stained with LIVE/DEAD® Viability/Cytotoxicity Kit (Themo Fisher: L3224). After T cell purification and tumor cell staining, we add T cells to tumor cells in 200 ul medium. Finally, the cytotoxicity were observed in In-Cell-Analyzer 2200 (GE) for 6 hours by real-time imaging and count for red signal of dead tumor cells. The RNA of T cells were collected after recording of images, and cytotoxicity genes expression, like IFNG, PRF1, GZMB, will be run on real-time qPCR. CD3 will be used as housekeeping gene.

I know the PHA will activate T cells and stimulate them proliferating via CD2 and calcium influx pathway. And I do observe obvious cytotoxicity in the preliminary experiments. Could anybody give me some advices to improve this method?  . 

I am generating Luciferase-expressing stable Tumor Cell Lines.

First i co-transfected the Lentiviral plasmid containing the luciferase gene with 293t cell and produce the virus. Than i transduce the tumor cell line with the susp collected from from 293t transfected cell.

How should i analyzed that my tumor cell line express the luciferase successfully? I study some papers for protocol , but their plasmid have GFP reporter gene, so its quit easy to detect. But mine plasmid don't have GFP or any other reporter gene.

thanks.

I'm trying to study NAD+ metabolism in cancer cells. I'd like to study the levels of NAD+, nicotinamide and nicotinamide mononucleotide (NMN). Assay kits for ATP and NAD+ are available but I haven't found any for NMN or nicotinamide. Any suggestions?

I'm interested in culturing solid primary tumor cells using trypsin. Does anyone have any good protocols or literature they could send my way please?

So far this is what I've found but I'd like to have a cheaper/more recent alternative

Thanks!

Hello,

I'm trying to get some mouse xenografts started for a gastric model that basically involves efficacy testing. What are some good human gastric cancer xenografts that give consistent tumors in nude mice with short latency?

Does the NO product stays inside the cell or is it excreted in the extracellular medium? I am not sure if I shoud add the reagent in the intact cell (adhered in the bottom of the 96-wells plate), take out the treated medium and put fresh one instead, or if I should keep the old medium ?

Im working on the targeting of NPs to specific tumor cell and to relase the payloads there.

I need the media for conditioning of my DCs. Also I wish to check for the expression of phosphorylated STAT 3. Can You please help me out with the duration of conditioning.

 I‘m studying a natural product's anti-tumor effect on melanoma. I was noticing it  triggered ER stress,  but at the same time, the autophagy related protein such as ATG5, ATG12 were down regulated. Also, the ratio of LC3-I and LC-II was elevated. As I know, the ER stress is usually considered as an inducer of autophagy. So I wondered if It possible to get such conclusion or do I need to do more experiments to prove that?

MG-63 cells, a line derived from an osteosarcoma, produce high yields of interferon after superinduction with polyinosinic acid polycytidylic acid, cycloheximide, and actinomycin D. Studies using MG-63 cells provide some important mechanistic clues concerning the details of the amplification process in tumors. Cells can also be transfected

Hi, I am planning to isolate single cells from human lung tumour tissue. I am wondering what is the best way to enzymatically degrade the tissue to isolate MOST of the cells (since the population of interest is very rare and I want to avoid false negatives when I do FACS analysis). I came across multiple collagenase cocktails which have been reported in literature, but not sure which is the best way forward. Which is the best collagenase to work with?

Any leads would be appreciated. I don't want to optimise too much since these are precious patient samples. Thank you! :)

I want to culture glioma tumour spheres/neurospheres from Gl261 adherent glioma culture.I am going to use a media having DMEM/F12 supplemented with N2,bFGF and EGF.Could anyone please suggest how much N2 I should add to my media?

Thanks in advance for your valuable input.

Hello there,

I am working to isolate breast cancer stem cells from MDA-MB-231 using CD44+/CD24low positive selection on magnetic separation (Miltenyi Biotech column). Has anybody used this kit for solid tumor cells and not hematopoietic cells that the protocol is based on? I reach a good enrichment but I end with very low number of cells to work with. Any suggestion is helpful.

I want to develop 4T1 tumor model. How many cells are needed to develop a subcutaneous tumor in Balb C mice?

Should I can use male Balb C mice in place of female?

If anyone have any clue about this, please guide me ASAP.

I've begun to establish some PDX lines that consistently passage through mice. At this point, I'd like to make a cell line from these PDX tissues. When I dissociate the tumor tissue and plate it I end up with a lot of mouse stroma that eventually takes over the culture. I'm hoping someone has a good protocol for deriving a purified human tumor line from PDX tissue. We have a Miltenyi MACS system but have not have success using their mouse cell depletion kits after tumor dissociation. At this point any alternative approach could help get over this hurdle. 

Thank you

Hey Guys,

I am about to set up a drug release experiment and it requires the pH of buffer to be tumorigenic to mimic tumors. I went through literature and it tells that people have used pH ranging 5-6.8. This got me confused a bit as in which buffer I need to make. So, could you please tell me what exactly is the pH of tumor cells and what should be used for drug release experiment?

Thanks.

I'm interested in identifying T-cell populations in mouse tumor sections. I can do IHC or IF and I have paraffin or frozen sections.

I induce tumor in mouse hindlimb using a very well known carcinogen and then at the onset of tumor initiation (visible bulge) I isolate the tumor, treat it with collagenase IV (0.2%, 2h) and plate the sieved cells in PLL-coated dishes. The cells are usually small and round and take 1-2 days to take their characteristic morphology, mostly fibroblast-like cell shapes. I have succeeded in isolating a mixed culture of cells using this protocol twice before but recently I repeated the same protocol thrice and failed in getting cells that take their characteristic morphology. I kept the cells for 5-7 days but they remained in their initial round phenotype. Several cells were adhered (I suspect its simply because of the PLL coated substratum), most cells were floating. I am completely amateur in primary cell culture and am unable to reason why I couldn't reproducethe same results with unchanged protocol, using which I could isolate cells 2-3 times before. I request for some suggestions.

We have a breast cancer mouse model from which we want to extract the tumour, dissociate in single cells and then remove all non-tumour cells to have a pure population of tumour cells to inject in NOD0SCID mice.

Does anyone knows how to remove non-tumour cells?

Specific markers for fibroblast and others non-tumour cells...

Does there is any drugs that can specific kill T cells without harm NK cells?

I suspect that my knockout tumor cells are more susceptible to granzyme-mediated killing.  Is there an in vitro assay to incubate tumor cells with granzyme (and perforin?) to induce cell death?  I thought this might be easier before attempting to incubate my tumor cells with isolated NKs or CTLs.

Any references or protocols would be greatly appreciated!

I want to draw the tumor clone evolution as follows, does anyone can help me which software i should use?

I am working on the effect of fenofibrate on the tumor cell metastasis by using Balb/c mice as the animal model. Now I have a problem which it's the fenofibrate is not soluble in water, so I used stock DMSO as a solvent. The required dose of FF was100mg/1000g, so I will need to prepare this dose by using a high amount of DMSO because the solubility of FF in DMSO IS 15mg/ml. AlL mice were severely tired after being  injected with this dose! Btw the control group also were died after being injected with DMSO alone !!!! any suggestions please!

Hi,

I am ex vivo activating CD8 T cells for an adoptive cell therapy model. I would like to freeze down batches of activated cells to use at later timepoints and in other assays. How well do the activated cells respond after freeze/thawing and how long should they be cultured after thawing before I look for cytokine production?

Thanks,

Hello everyone, I am starting to work on extracellular vesicles (EVs) so recently I did an exosome isolation from cell culture media of neuroglioma cells by using ultracentrifugation protocol. I checked the EVs size with Nanosight and obtained several pics: two major at 165 and 215 nm and two other but smaller at 355 and 535 nm (might be aggregates). Does anyone know if this profile looks like something I could expect or is that too much high for a classical EVs profile? Usually the major pic is around 100nm in accordance with literature data but it seems to be the expected profile from exosomes (a small portion of EVs from cells). Is there any cut-off I should consider more related to all type of EVs?

Dear all,

I constructed a mutant 4T1 cell line using CRISPR and implanted it into the 4th mammary gland of BALB/c recently. The tumor grows well at the same rate as WT in the first 12 days but 2 weeks later some of the CRISPR-modified tumor regressed while some remains small about 4~5mm long. I have some guesses but I really need you guy's assitance to help me out.

1. Maybe it's just a good thing, a phenotype. The presence of my gene ruins the immunosurvilence so the mutant cells are attacked and ruled out by the immune cells.

2. The contamination of Mycoplasm. But I think the contaminated cells cannot grow into a tumor at all. I don't have so much experience, so any information about this is appreciated.

3. The loss of cell viability. You know, it takes almost 2 months to finish the entire work of CRISPR. So I am afraid the 4T1 invasiveness will decrease. But for the same reason mentioned in 2, I think if viability is an issue the tumor will not appear at all. I need you guy's help to give me any experiences about this.

Thanks in advance for all your time and kind help, guys!

Dear all, 

I have tried to use 40:80% Percoll to isolate tumor-infiltrating lymphocytes from tumor tissue of CT26 BALB/c mice model. But when I used this method after collagenase digestion and centrifugation (300rpm, 25 min), no separated parts of any cell types were found. The tumor cells were still mixed in 40% Percoll layer which it should truly be sedimented at the bottom of the tube. I have tried to used the other concentrations of Percoll (75%, 70% and 65%) instead of 80% but it showed the same results. Does anyone have suggestions or experiences about this? I will very appreciate it.

Thanks!!       

Hi all,

I have seeded and have been expanding a set of Organoids over the last 3 weeks. The Organoids are derived from Melanoma tumor. The method I have been using is to dissolve the matrigel in Cell recovery solution for 1-2 hours , spin down, remove Matrigel with Cell recovery solution, wash with PBS, add enough Matrigel for 3 times as many wells that were harvested, mix mechanically, distribute to the new wells. The main problem I have run into is that I am getting several wells that have very little cells transferred to them along with a few that have no cells at all. Half of the wells will have the majority of cells. Short of cutting back on the number of wells I expand to or painstakingly mechanically removing single colonies during transfer into the wells, I am at a loss. I know that if I can break up the masses that have developed over the weeks without damaging the cells I have a higher likelihood of expanding the cells to more wells. 

Does anyone have experience with this? Can you share a method you used?

Thanks.

I would like to establish a immunotherapy model by co-culturing human tumor cell lines with HLA-matched T cells. In particular I am interested in non small cell lung cancer (NSCLC) cell line Calu-1.The HLA phenotype for Calu-1 cells is as follows: HLA A10, A11, B15, Bw35. I need at least partially matched HLA-phenotype, i.e. matching the A-subclass. 

Is there any company that provide PBMCs which are characterized for HLA phenotype? 

Thanks,

Alessia 

Hi, guys, I know it's not easy to culture primary cells because they have limited cell division.

But for us, we are only able to passage cells once or even worse.

1) Cells are either growing slowly or enter senescence easily.

2) After cell passaging, the morphology also changes .

We are culturing ovarian cancer cells, and use DMEM+EGF+FGF+B27 with 0~2% FBS.

I also attached some pictures here from your reference.

Does anyone  know what happen? and how to modify the culture condition to make the cells in proliferation and also maintain the morphology?

Thanks a lot!!

I am setting up an experiment where I use magnetic beads to positively select for CD4+ T cells from a C57BL/6 mouse (with a tumour, either untreated or with treatment) spleen, followed by expanding the population. I then want to do a direct coculture with a cancer cell line, where I will conduct an ELISA on the supernatant to assess cytokine production (IL-12 etc.). My question is, by expanding the CD4+ T cells ex vivo (using antiCD3/antiCD28 and IL-2), would I be selectively expanding a specific subset (Th1 over Tregs for example), thereby skewing my results? If so, how might I go about avoiding that?

Dear all,

I'm looking for a tumor cell line expressing OVA protein on the cell surface so that anti-OVA antibody in sera can bind to the cell through OVA protein.

Does anyone has that cell line or know where can I get it from?

Thank you very much!

So my question is, whether somebody has experience with a similar set-up as described below (co-culturing target & effector cells for several days and then look at the killing efficacy of the allospecific effector cells) and if so, how he/she sets up the Assay and what control he/she uses.

So what I want to look at is:

I co-culture 40.000 irradiated tumor cells (suspension cells) for 5 days with 200.000 effector cells and administer different treatments.

After those 5 days (when I have allospecific cells) I wash the cells twice and I add again 40.000 tumors cells (not irradiated).

After 16 h I aquire the data.

As a maximum release control I use 40.000 tumor cells and kill them with 2 % Triton, and to be honest for the spontaneous release I don't really know what to use... 

So far I didn't get a stable readout and often the killing comes near 100 % or even more in comparison to the Triton wells, which of course makes sense, since in the other wells there are also dead effector cells and maybe some of the tumor cells from the co-culturing weren't killed until then...

I'm really thankful for any help!

Hello, I need to generate tumors in nude mice with the cell lines SK MEL 28 (melanoma) and MDA MB 231 (breast cancer), both human tumoral cell lines. How many cells do i have to inject intradermically?

Thanks, 

Martin

Can a tumour secrete a product but not expressing the protein?

Thanks

Hi all,

Having a ton of trouble infecting my primary mouse T cells from spleen with lentivirus. I regularly am getting 5-10% infection rate and many people seem to say this is within the "norm" but I would love to just get a little bit higher. I've tried everything - retronectin, protamine sulfate, dextran, polybrene, IL media - but nothing seems to get the rate up. Does anyone have any tips or seem to think this is going to be it for me?

Thanks!

Good Morning.

I should evaluate the expression of membrane protein in a luminal cancer cell lines (HER2-negative, ER-Positive) derived by spontaneous tumor growth in transgenic or not mouse.

Do you have an idea if such cell line exists?

Thank you very much

Kind Regard

Lorenzo

I'm looking for one Ab targeting CD44-HA interaction and one that doesn't. The aim is to use the Abs to disrupt CD44 adhesion/signaling in an in vitro tumor cell culture, eventually leading to further in vivo studies adminiestering Abs to mice. Alternatively, how feasible would be the use of hyaluronidases or HA oligosaccharides to inhibit CD44-HA interaction? In the case of HA oligosaccharides, which one(s) would be best as inhibitors?

I am having difficulty extracting RNA from fresh frozen tumor samples. I am having good extractions on about 10/18 samples. I am following the protocol exactly. I have recently noticed- when spinning the sample down after breaking up the tissue, I see a pellet, but I also see a filmy substance floating around the top of the supernatant. These samples are having 0 yield. Does anyone know what this is? Or advice on using this kit? 

how to monitor tumor propagation in lung after inoculation of cell lines in the lung. Is there any simple and reliable method by which it can be check the gradual increase in the size of tumor in lung. if any than please share the methods with references..... 

I was looking in the literature for prostate cancer C4-2 cells (CRPC) mouse xenografts that used enzalutamide, but I wasn't able to find any such papers. Could any of you help me with this? Thanks in advance. 

We tried the model following the the current protocols in immunology: The TRAMP mouse as a model for prostate cancer. Unable to get tumors growing.

hepg2 cells looked floated and did not attach after 48 hours of revival process. 

If so what factors do they need?

We need a clear protocol to induce liver cancer in rats, in a shorter time and less expensive. 

Thank you in advance for help

We are interested in imaging co-cultures of tumor cells and immune cells, but would like to culture them within the same dish. Is this possible? Or is there a fundamental reason all the co-culture papers use transwells?

I want to do an experiment where I take conditioned media from one plate of cells and and add it to a plate of cells with a gene knockout and see if there are changes in phenotype.  Can someone run through a protocol on how to do this.  Do I add serum in the plate while I am conditioning the media but no serum to the plate of cells for which I will be adding the conditioned media to or no serum at all? These are lung tumor cells which normally take RPMI with 10% FBS.

I want to find the migration difference between transfected and non-transfected tumor cell lines, I'm considering three brand migration assays: Falcon, Corning and BD Biosciences. For migration assays, the membrane pore should be 8um. For Corning, there are different membrane types, like polyester (PET), polycarbonate (PC) and collagen-coated (PTFE). Besides, it seems there are some relationship among Falon, Corning and BD Biosciences, it makes me confused. Briefly, who can tell me the differences among these different membrane. It would be appreciated very much?

I recently ran an experiment where I divided a mouse tumor into several wells of a 96-well plate. Each well was stained with a different panel of antibodies. In only one of the wells, I got the strange pattern you see on the far right, where my events formed a distinct diagonal line with a slope much smaller than expected. If I gate on the events, they are abnormal in the other plots as well. Any help you can give would be great, thank you!! 

I work on different cancer cell lines (breast, liver, colon) , contamination occurs either the flask becomes turbid or fungus grows on the inside off the flask, i clean the incubator with chlorine and 70% alcohol, i use antimycotic and filter the complete media how shall i get rid of that aggressive contamination please?

I work with B16-F10 melanoma cells, and I have a huge problem with infections. I think, that this infection is caused by some kind of yeast, but I'm not sure. Have anybody an idea? Thank you!

i have treated a tumor cell line using a plant extract and terminated the treatment at different times (eg: 1 hour, 2 hours, 4 hrs and so on) and estimated some enzyme activities and observed that the enzymes decreased up to a certain time, increased again and then decreased again....  how do i interpret this and what exactly is happening??

thank you

Hi!

I trying to block bcl-2 in bcl-2-overexpressing cell culture model. In my conditions ABT take effect at 2 mkM - cells turns apoptosis and dead. But ABT-199 active at nanomoles. What concentration you use and which way to confirm bcl-2 inhibition?

Thanks!

I have prepared tumour spheroids with U87 MG cells and I am trying to get fluorescence images using confocal microscope. However when I placed tumour spheroids between cover slips and microscope slides, I think they get squeezed and I lose the 3D shape of the spheroids. Do you have any suggestion to prevent this? Thank you.

These cell lines along with available xenografts and other factors along with other biomarkers ( if you have any it would be appreciated)  will be used to build a preclinical model for high risk Wilms tumors.  The development of assays specific to this model will result in the ability to do novel cell line and animal testing which could potentially lead to clinical trials with new compounds for children with these disease.

i want to count gamma h2ax foci in the tumoral cells,which software is suitable?

i want to choose the concentration of the drug that has the most efficacy, is flow cytometry of cell cycle a suitable test or not?

Hello everyone,

our problem is non formation of tumors in xenograft expt. We are using female nude mice.

We injected HO15.19 (rat1 a) cells expressing Myc as follows:

1. First female nude mouse: we injected HO15.19 (rat1 a) cells expressing WT Myc : 2 million cells+matrigel into one flank and 3 million cells in PBS into another flank.

2. Second female mouse: we injected HO15.19 (rat1 a) cells expressing mutant Myc : 1 million cells in matrigel into one flank and 5 million cells in PBS on another flank.

It has been 22 days and we still see no trace of tumors. Can anyone help troubleshoot the problem. I am a novice in the field and had not done tumorigenesis studies before?

I try to figure out some easy method to transfer spheroids from hanging drops from a petri dish lid into 96-well plates. So I had the idea to generate the tumor spheroids directly on the lid of a 96 well-plate directly instead of generating them on petri dish lids and to centrifuge the plate after spheroid initiation. Does that work to centrifuge the spheroids from the lid directly into the well? 

I generated them with hanging-drops and transferred them into 48 well-plates for further cultivation. But after 8 days they didn't look really solid and compact anymore. And when I tried to transfer them into a falcon tube for other experiments, they actually dissolved during pipetting.

I am culturing liver cancer cell line in serum free tumour sphere medium (DMEM/F12 + EGF + bFGF) and culturing them for at least 7 days to enrich for cancer stem cells. Do I need to change the medium everyday?

I have a concern that this will disturb the sphere formation as I will need to centrifuge and I am afraid that during the resuspension in fresh media will affect the spheres. I am culturing them in 6-well ultra low plate by the way.

I aim to perform a study about circulating tumor cells in colorectal cancer and to culture CTC s

How can culture cells from fresh tissue of breast cancer?

Hello everyone,

Does anyone know how I can isolate and transfer a spheroid from one plate to another one? 

how can I wash a spheroid with PBS (e.g. Can i use centrifugation method? and how can I cut the spheroids to see their cross-sections?

Thank you.

Looking at tumoursphere formation in a paper which uses LM2 (triple-negative) and MCF7 (oestrogen-receptor positive) cell lines. Control comparisons show a much lower tumoursphere formation - is this because the culture medium did not contain any oestrogen supplementation? No experience culturing cells / whether this is something you can accept as an inherent difference between the cells?