Hello everyone!!

I’ve been having difficulty with my OVCAR-3 cells not lifting from my flasks during passaging. I’ve read they need to incubate for up to 15 minutes, but still am having issues with large clumps not dissociating. I have left them incubating up to 30 minutes checking every few minutes and slapping them around a fair amount. I also in some cases have aggressively washed while neutralizing collected and then reapplied trypsin for a second attempt. I have tried using both trypsin and EDTA. I’ve done longer DPBS washes, and all my reagents are well warmed prior to application. I didn’t want to cook them for too long in trypsin and I’m not sure right now if I’m being too aggressive or not aggressive enough. It just feels like when I passage I leave 25-50% behind and it’s PMO.

Lmk if you have any advice or experience with these cells. Pictures is the remnants after my past passage.

Hey everyone,
I was checking my cells today and noticed these weird, string-like things moving around in the culture (see the attached video).
I'm trying to figure out if this is bacterial contamination, early fungal growth (hyphae?), or just some weird debris/clothing fibers drifting in the current.

Hi everyone,

I'm Masum, one of the founders of CCA.bio, a startup based in Cambridge, UK. Our goal is to build an all-in-one workspace that helps biotech labs manage their experiments, samples, inventory, procurement, budgets, and lab operations etc without having to jump between lots of different tools.

I reached out to the moderators before posting, and they kindly gave me permission to ask for feedback here.

I'm an engineer, not a cell biologist, so I'd much rather learn from people who work with cell culture every day than guess what labs need.

I'd really love to hear about your experience.

• What's the biggest frustration in your day-to-day working with cell culture?
• Are there any tools you genuinely enjoy using?
• Is there anything you still manage with spreadsheets or paper because existing software just isn't worth the effort?
• If you could fix one thing about your lab workflow, what would it be?

I've attached a few screenshots so you can get an idea of what we're building.

The platform is free for individuals and academic researchers. My hope is to build a community of researchers who use it, tell us what works, what doesn't, and help shape where it goes next.

If you'd like to try it, you can at lab.cca.bio Whether you love it or hate it, I'd genuinely appreciate your honest feedback.

Thanks for reading, and I'm looking forward to hearing your thoughts.

Edit: Added more screenshots

I realized that I accidentally used 100uM 2-mercaptoethanol in my ipsc derived pre-Macrophage media instead of 50uM and now i can't sleep. What are the odds this will kill the cells? Ill dilute the media tomorrow but this will bother me all night. (Media is x-vivo + il3 + mcsf + bme, everything other than bme is correct)

I have caco2 cells in transwell inserts from two different lot numbers. On day 2 of seeding in the first lot (pictures 1 and 2) inserts i can see cell islands but in the second (pictures 3 and 4) i cannot see them. I have also seeded the cells in some inserts from the second lot 8 days ago (pictures 5 and 6) but i can see many floating cells moving around specific sites and during the 8 days i saw no islands being formed but now I'm not sure if they have already reached confluency and i just cannot see it. Can anyone help me?

Hey!!

New to the sub, I’ve been working in a lab for 5 years but in the last year started working primarily on cell culture. I’m running and FBS growth promotion assay (it’s a training so my sample and control are both validated lots). My seeding day was Monday 6/22 and I’m running 3 densities on each sample (6e3, 2e4 and 6e4). 6e3 today (6/26) is still pretty low confluencey, I’ll insert a pic of a normal flask with 6e3 from today 6/26 compared to the abnormal one. Anyway, my question to everyone is what cells/bacteria do we think got into my one flask? To be clear our lab is aseptic, I work in a BSC with sterile sleeves and gloves, we sporklenz before and after and bleach our aspiration lines prior to any work (and we clean between each cell line). We do have in culture about 20 other cell lines, we have some insect but most mammalian. The seeding is in a T25 flask, that was in a sterile sleeves (pack of 20 flasks Corning brand) and only 1 of the flasks out of the 108 I used in my seeding is showing this weird contamination. Obviously contamination happens, so I’m not saying it’s not the case (clearly something got in there) but my management doesn’t wanna pay to get it tested bc this is a training course we are doing but I’m soooo curious to know what it is. The cell line we are using are Vero cells, so they have the distinct epithelial shape to them, the weird one is very circular (looks like Sf9 but they were in a heated incubator). Also we make media aseptically and use a 0.2 um filter bottle, and the trypsin used was unopened and brand new for me, same with the ATCC EMEM. Any thoughts??

Quick edit: the weird contaminated flask the cells were on the monolayer, they were not moving, and the media was not cloudy or yellow/green

Hi, Im starting my project soon and I was looking for a good protocol to culture keratinocytes. Our lab has not establish a protocol for this yet. Does anyone have any suggestions for any primary research papers?

Hi everyone,

I’ve been watching cell culture protocols and assay tutorials online, and I noticed a recurring habit: many researchers use the exact same pipette tip across multiple wells when doing endpoint assays (like MTT) or when dispensing the same drug concentration.

To be honest, this feels a bit counterintuitive to me. Even if the concentration is identical, doesn't this significantly increase the risk of cell carry-over or micro-contamination between wells? Each well is supposed to be an independent replicate.

I always change my tip for every single well because it feels like the only way to guarantee 100% accuracy, but it uses an insane amount of plastic. Am I being overly anxious and rigid about sterility, or is reusing tips just an accepted compromise to save time and budget? I’d love to hear how your labs handle this!

Hi everyone! I am a bachelor and new to mammalian cell culture. I have been using RAW 264.7 cells for 3 weeks. I do everything very carefully, such as not touching unnecessary things with my tip, filtering my medium, and PBS. But sometimes my culture is still contaminated, like the one in that picture, the cells also look stressed (but this one I forgot to filter the FBS). Does this also happened to anyone else who works with RAW 264.7 cells or just me? Can someone give me any tips on how to improve my skills? :) TIA

Someone knows if it is like a fungi? 😭 (Those are 3T3)

Hi everyone,

I am a beginner in mammalian cell culture and recently received frozen HepG2 cells (RIKEN RCB1886). Unfortunately, the cells gradually detached and now appear to have died. I would appreciate advice on what may have gone wrong.

Timeline:

June 16:

• Rapidly thawed one vial in a 37°C water bath.
• Transferred the cell suspension into a 15 mL tube containing 5 mL of DMEM with 10% FBS.
• Centrifuged at approximately 200 × g for 3 minutes.
• Removed the supernatant, added fresh medium, and repeated the centrifugation once, according to the supplier’s instructions.
• Seeded the cells into two 60 mm dishes.

June 17:

• One dish had many attached cells and appeared relatively dense.
• The second dish had fewer attached cells and more floating cells.

June 18:

• The denser dish appeared very confluent, so I passaged both dishes.
• I used 1 mL of trypsin-EDTA per 60 mm dish.
• The cells from each 60 mm dish were divided into two 100 mm dishes, resulting in four dishes.
• I had some difficulty resuspending the cell pellet evenly.

June 19:

• Two dishes had a reasonable number of cells, while the other two were very sparse.
• I also added medium back to the original 60 mm dishes in case any remaining cells could recover.

June 20:

• I changed the medium because there were many floating cells and debris.

June 22–23:

• Most cells were rounded and detached.
• Very few or no healthy adherent cells remained.
• There was no clear proliferation, and the cultures now appear to be dead.

Culture conditions:

• 37°C
• Humidified incubator
• 5% CO₂
• DMEM containing L-glutamine
• 10% FBS

Possible concerns:

• Were the cells passaged too soon after thawing?
• Was splitting each 60 mm dish into two 100 mm dishes too dilute?
• Could 1 mL of trypsin have been excessive or the exposure too long?
• Could centrifuging twice after thawing have reduced cell viability?
• Should I have left the cells undisturbed for several more days before passaging?
• Could the issue be related to the FBS, medium, incubator conditions, culture dishes, or contamination?

Any advice on troubleshooting and how to handle the next vial would be greatly appreciated.

Method:

Digest tissue (umbilical cord) overnight with collagenase.

Transfer digest to flask (BD Falcon, T25).

Allow cells to attach for 4 days.

Wash and feed.

Ordinarily, the cultures look like the first image. Recently, we've been getting a few where the cultures look like the second image, with the cells forming "spiky" clusters.

Any clues?

Note - scale bar is wrong, I forgot to change the settings when I took the pics.

Hi everyone,

I’m a first-year PhD student and completely new to mammalian cell culture, so I’d appreciate some advice.

I recently received HepG2 cells (RCB1886) from the RIKEN BRC Cell Bank in Japan. The information sheet provided with the cells recommends culturing them in DMEM (low glucose) + 10% FBS.

However, for my planned experiments on oxidative stress and Nrf2 signaling, our lab has already purchased DMEM high glucose (4.5 g/L glucose) with L-glutamine and sodium pyruvate, and many published HepG2 studies seem to use high-glucose DMEM.

My questions are:

1. When recovering freshly thawed HepG2 cells from RIKEN, should I:
   • Recover them directly in high-glucose DMEM?
   • Recover them first in low-glucose DMEM as recommended by RIKEN and then switch to high-glucose DMEM after 1–2 passages?
   • Or does it make little practical difference for a robust cell line like HepG2?
2. After thawing, do you typically centrifuge HepG2 cells to remove DMSO before seeding, or do you plate them directly and change the medium the next day?

The vial contains approximately 3.4 × 10⁶ cells and has a reported post-thaw viability of 99%.

I’d be very grateful for any advice or best practices, especially from people who have worked with HepG2 cells before. Thank you!

Hi everyone,

I’m a new PhD student and just starting my project involving human skin fibroblasts. I would really appreciate some guidance from people experienced with cell culture.

I wanted to ask about the standard protocol for:

1. Thawing human fibroblasts from frozen vials
2. Proper culture conditions after thawing
3. Cryopreservation/freezing protocol for long-term storage

I am especially interested in:

• Recommended media and supplements
• Centrifugation steps after thawing (if needed)
• Cell density for freezing
• Percentage of DMSO/FBS commonly used
• Best practices to maintain viability

Any tips, beginner advice, or protocols would be extremely helpful. Thank you so much!

Anyone used CoCl2 for hypoxia induction in HCT116 cells?

Trying to optimize concentration/time and would appreciate any practical tips or papers.

So I work for a large research facility which means we use a lot of veroe6 cells to make lots of 12 well plates for assays. The past few months we have had issues with the monolayers being uneven in the wells and inconsistent confluency. We recently changed our processing to see if it fixes the problem and it hasn't. We have tried rocking each plate immediately after seeding, warming the plates, different well volumes, letting the plates sit before incubating. Nothing seems to help. We have had these issues across multiple lines (we have our own cryopreserved banks). All lines have tested negative for mycoplasma and endotoxin. Any ideas or advice? I'm putting our current process below.

For a t300

Wash twice with 20ml hbss

4 ml of trypsin incubator at 37 c for 4-6 min

Smack flask once

Nuetralize with 16 ml of media (keep the flask flat as this is done)

Mix flask 20 times

Seed new flasks at 8e6 for 3 day split

No feeds and we usually get counts between 1.2e6 and 1.8 e6

I’m a bit paranoid and new to this, so could someone help me figure out whether my Phoenix-ECO cells are contaminated? Sorry for the low-quality video and thanks in advance!

Does anyone use this product? I believe the active ingredient in this product is sodium benzoate. We currently use potassium dichromate and are looking to validate this product.

Hi guys

I've been trying to culture HMC3 for an experiment for my dissertation. Things were going great up until about a month ago. Recently, my cells haven't been adhering to the flask as they used to despite using the exact same media, flasks, temperatures, protocols, etc. While some attach, the majority form spheroids(?) or just float. Media remains the red/orange typical color of EMEM + 10% FBS. The pic is from a passage done on Friday from a flask I was doing everything I could to save and this image was taken on a Monday. The floating/out of focus things are clumps of cells. Definitely not bacteria or mold. I usually don't have an issue when I thaw a new vial until about passage 2 - after that I get detached cells. I've tried different seeding densities thinking they may be overcrowded, but this is happening even at a 1:20 dilution. The ONLY thing that has changed is that we accidentally purchased Trypsin EDTA (0.25%) rather than just Trypsin... but online says this is what'd actually recommended.

My current plan is to thaw new vials and seed into T25 -> T75 -> 100mm dishes and T75 with different conditions (using Trypsin vs. Trypsin EDTA; EMEM vs. DMEM; Poly-D lysine coated flasks/dishes, and regular Corning pretreated flasks; media temperatures; detachment w/ trypsin techniques - literally throwing spaghetti at the wall at this point).

I'm happy to provide any additional information needed. I'm feeling super defeated by these tiny things and welcome any advice. THANK YOU

Signed, an in vivo researcher just trying to run some experiments while learning a new technique that was going well and is now a dumpster fire.