This is something that I have examined across time. My experience is with the MAVER-1 and JeKo-1 lines, I do not have Mino, Granta-519 or REC-1 cells.

If you received these lines from ATCC, the culture information stresses the importance of keeping the cell concentrations low - if I recall correctly, the number they cited for JeKo-1 was less than 2 million per ml, which JeKo-1 can readily hit, but the number was even lower for MAVER-1. When the cell concentration exceeded the threshold, what happened in my experience was very striking. Many of the cells would die off, and the cultures would look terrible. You can recover the cells by spinning them down and resuspending them in fresh culture medium, but it took a week or two to recover.

I was examining the cells by flow cytometry and discovered that both of these lines express CD178 - also known as Fas Ligand - at extremely high levels - 10 fold brighter than CD45. As the cell concentrations increased, there was also an increase in expression of Fas/CD95. So when the cell concentration hits the threshold - which seems to be different in MAVER-1 vs JeKo-1 - the culture die-off occurs. I tried to inhibit this with anti-Fas antibody, but the results were not impressive.

One way to avoid this is to subculture the cells frequently. I was splitting the cells twice a week instead of once a week. Eventually, the cells adapt to that, and the die-offs cease to occur. This correlates with a marked diminution of CD178 expression - now the same as CD45. This has happened with both MAVER-1 and JeKo-1. Due to a freezer failure, I have lost my stocks of the lines when they were CD178 bright, and new stocks from ATCC seem also to have been culture adapted. They do not carry the warning about cell concentrations exceeding a certain level.

The high level of CD178 expression likely points to the biological function of mantle cells - deletion of self-reactive T cells. In lymph node and spleen, during the immune response, activated T cells migrate through the mantle and would encounter mantle cells. It is also known that class-II self reactive T cells are deleted in the thymus by CD5+ B cells in mice.

I was planning to write these results up and submit a manuscript, but without CD178-bright cells, I am at an impasse.

How often do you split these cells and at which confluency?

it is critical not to culture these cells to high confluency >1-1.5*10^6.

I"v cultured REC-1 and Jeko-1 cells in RPMI medium supplemented 10% or 20% FBS, respectively. I used to split these cells 2-3 times a week in culture: fresh media ratio of 1:3 in the case of REC-1 and 1:6-10 in the case of Jeko-1! and didn't experience any problems with them.

However, I do recommend using fresh cultures of MCL cells every ~60-70 days. It's well known that MCL cells are genetically unstable because of the aberrant regulation of their cell cycle, DNA damage pathways, etc..., constantly generate new mutations and other genetic alternations on the genomic levels.

check out this review: Article Mantle cell lymphoma: Biology, pathogenesis, and the molecul...

Thank you Gary Lee Gilmore Aladin Samara for the very interesting answers!

I noticed that Jeko-1 and Mino have an higher proliferation rate and they maintain a good viability for long period without suffering for high confluence. On the other side REC-1 and MAVER-1 are more sensitive to confluence and time. Have you noticed that?

I split cells 2 times/week with a confluency 0.8-0.9*10^6/mL. I'm not sure I can maintain a ratio of 1:3 for my REC-1 culture because of the low proliferation rate. Do you have any suggestion for that?

Do you think that the addition of Hepes, non-essential amino acids or Na pyruvate can improve the cell culture?

Thank you very much

JeKo-1 definitely proliferates faster than MAVER-1, often acidifying the medium to yellowish orange, while MAVER-1 does not. I have no experience with either REC-1 or Mino.

Hepes is a buffer. RPMI medium only has bicarbonate buffer, so Hepes helps hold the pH near optimum for cell growth. It is a component of IMDM. I do not use it for MAVER-1 or JeKo-1 culture, but it could help.

At low cell densities, cells leak out small amino acids like glycine and alanine, and either Na pyurvate or non-essential amino acids compensate for that - pyruvate can be transaminated into alanine, which can be converted to glycine or carboxylated to aspartic acid, which can be amidated to asparagine.

I have used Na pyruvate to help MAVER-1 come out of cryopreservation. I typically do not use it for continuous culture, but it could also help.

You can try adding β-mercaptoethanol (BME).

Luca Pagliaro

I have experience working with other cell lines VERO, HeLa, HUT , PBMCs etc., not the one you have mentioned.

However, I felt you need to closely work on the cell concentration !!

Do you have any standard procedure to follow?

L glutamine needs to be added in some media that helped in resolving the viability issue we were facing. the concentration is recommended as per ATCC dissolve in distilled water and millipore filter can be added to the media bottle.

i logged in thopguh my account but my seniors name is coming am Michael Durairaj

I am actually having this problem with Mino cells. I could observe that low seeding concentration negatively affects them and decreases proliferation rate. But what I couldn´t determine if the best concentration for keeping them in culture, and for 48 h-experiment. I consider that both the concentration of the starting culture and cell seeding directly influences the response to my treatment. Please, let me know if someone has some advice (I tried with 100000 to 350000 cells/ml and it´s not working very well). Thanks in advance!

María Julia Lamberti

If you like then come to my inbox !!

To Maria Julia Lamberti - when cells do not proliferate at low seeding densities, it is often because cells tend to leak out small amino acids like glycine and alanine, and if the cell density is too low, they cannot grow properly. This can usually be prevented by adding 1 mM sodium pyruvate to the medium, which can be readily converted into alanine and glycine from alanine. I add pyrvate to my medium whenever I am doing limiting dilution or thawing a cell line from cryostorage.

Rarely, pyruvate doesn't solve the problem, in which case I use non-essential amino acids.

https://www.atcc.org/products/all/30-2214.aspx

L-Glutamine is an essential amino acid required by virtually all mammalian and insect cells grown in culture.

L-Glutamine is also more labile in cell culture solutions than other amino acids. The rate and extent of L-glutamine degradation are related to storage temperatures, age of the product, and pH.

Storage Conditions - Store L-glutamine solution frozen at -20°C or colder. Avoid repeated freeze-thaws by dispensing and storing in aliquots. after dissolving and miilipore filter and add to the media.@Dr B P Srinivas had identified the issue @IVRI SRC bangalore, while doing his PhD.

L-glutamine degradation results in the build-up of ammonia which can have a deleterious effect on some cell lines. Use caution when adding more L-glutamine than called for in the original medium formulation (2 to 4 mM is standard). For most cell lines, ammonia toxicity is more critical for cell viability than L-glutamine limitation.

Added as a supplement to cell culture media. L-Glutamine is an essential amino acid required by virtually all mammalian and insect cells grown in culture. L-Glutamine is also more labile in cell culture solutions than other amino acids. The rate and extent of L-glutamine degradation are related to storage temperatures, age of the product, and pH.

All cell culture media are formulated to contain appropriate amounts of L-glutamine to support cell growth well beyond the expiration date of the media. However, if L-glutamine is suspected to be a limiting factor during cell culture, a simple test of 'spiking' the medium with a small amount of L-glutamine will determine whether or not more is required. Simply add a small amount of L-glutamine (~ 2 mM final concentration) to the culture medium. If the cell growth rate increases, L-glutamine is most likely deficient and more should be added.