Hi Shabbir. Our lab does some work with HIT-T15 cells and we grow them in tissue culture treated flasks. When we plate them, we also use the tissue culture treated dishes.

Also, when we revive liquid nitrogen preserved HIT-T15 cells, we have them in 11.1mM glucose RPMI for a week before transferring them to their original appropriate glucose concentration RPMI.

Thanks Tsehay, for valuable information.

I would like to ask two things.

1. How do you come to know that cryopreserved HIT-T15 cells will have to incubate in the presence of 11.mM glucose concentration media. i mean do you have some sort of informative literature/article, if you have then please share it.

2. The RPMI media formulation which i m using is mentioned in my previous post, and this media is 2gm/l glucose. Sigma Cat# R0883. so should i add 2.5gm more glucose in one liter of media. then its concentration will be 4.5gm/l glucose.

This media without adding glucose in it, will serve as 11.1mM glucose concentration media. i will repeat it like this.

Thanks Tsehay once again, and for all those who read it.....

Shabbir,

I'm sure I can find papers that have done 11.1mM glucose to resuscitate cryopreserved HIT cells but I cannot say as to why that concentration exactly, other than its the way our lab has done it for a long while. I'll have my supervisor, who has thought me everything about HIT cells, take a look and see if she can respond to you on here.

Good luck

Hello Shabbir,

I have been working with this cell line for over 20 years, so I may be able to help.

We have done research with HIT-T15 cells from passage 60+ up through 100+ and use RPMI 1640 (with Hepes or without) supplemented with 10% FBS. We culture under standard conditions (37 C and 5% CO2). We don't use antibiotics, but if you wish to try without, I recommend making the switch after a split and freeze back, keeping at least one flask and several cryovials with antibiotics in case you have some kind of low level contamination. I have never added BME to HIT cells and do not know what sort of effect this will have on them. It is routinely used in INS-1 cell culture, though. We never have used F12, but we received our cells from Dr. Santerre, not ATCC.

The RPMI I use is Invitrogen's #11879 (no Hepes) or #22400 (with Hepes), but any brand should work. I even used to make my own from scratch before (-) glucose RPMI was readily available.

Glucose is 11.1 mM if you want to study glucotoxic cells but we also maintain cells at 0.8 mM long term for preserving insulin secretion patterns. The glucose dose response curve is left shifted, so 0.8 mM is quite enough and will stimulate insulin secretion, but cells should be subcultured at 11.1 mM if you want to study insulin secretion in these cells. It is necessary to rev up the insulin production.

I use Corning flasks and dishes and have found that the cells, after being grown for many passages in one type of tissue culture plate can grow more slowly on a different brand. I have never had a complete lack of growth and do not supplement my media other than the FBS. Note that for low glucose culture, you will need to account for the glucose in your FBS, which can vary a bit.

Could you share with me your source of cells and what passage you are working with? They do grow more slowly at low passage and more quickly at higher passages. After two weeks in culture in our P70 cells, they should be doubling nearly daily. We routinely will plate 5 million in a T75 flask and have 30 million in seven days. A weekly split at P70 is going to be ~1:4, but by P90 one may need to use 1:10 if not counting the cells.Also, what is your general plan, that is, what type of experiments will you do? Secretion, RNA harvest. protein harvest for western, PCR?

If you are interested, I can share my protocols for routine handling of these cells, but it will be helpful to know in general what techniques you intend to use with these cells.

Here is a link to some of our work.

http://diabetes.diabetesjournals.org/content/47/6/900.full.pdf

To answer your question to Tsehay regarding glucose concentration in HIT-T15 cells when thawing: We sometimes do long term cultures at 0.8 mM glucose. If we freeze these cells back and then wish to wake them up again for more experiments, we must wake them up in 11.1 mM glucose for a few days or up to a week before putting them back into 0.8 mM. If you wake them up in low glucose, you will lose a lot of cells. I do not believe that we explicitly mentioned this in our papers, but if a new person in the lab forgets or was not told, it is apparent when the low glucose cells are thawed out and don't survive well. We have an older paper about the effects of various culture conditions on these cells - JCI 90:320-325, 1992.

RPMI 1640 is 2 g/L glucose, which is equivalent to 11.1 mM. I would not add more than 2 g/L glucose to the culture media unless doing so is part of your research design, the cells don't need it and lose the ability to secrete insulin when maintained at 11.1 mM.

Thanks Elizabeth Oseid, for your detailed answer,

Sure, I will be thankful to you if you share your lab protocol, which I need.

We run semi dry western blotting for the target proteins. We worked on C2C12 cells on project of inhibitory function of Ser 357 Phosophorylation of IRS-1 in insulin signal transduction. Generally, we are doing mechanistic studies.

But for Hit-T15 cells, I did not run a single experiment. I am just trying to revive it, after finishing my MS course work. We have decided in case of Hit-T15 cells to use natural and synthetic antidiabetic compounds to see the effects on beta cell growth and survival, by analyzing gene as well as protein expression. We got Hit-T15 cells from ATCC, passage no was not mention on the vial. So, I considered it a first passage, however I login to ATCC, and I wrote a lot number in ATCC, in order to see the specific passage #. I will share it with you.

I have already added the BME in RPMI-1640 medium, and I think this might be one of the reason.

Yes, you are right, that I should not add more glucose, and I did not add it.

Once again, Thanks so much.

If you are culturing in regular RPMI 1640 with 10% FBS, then for western, you should be able to plate 5-10 million cells in a 100 cm dish and then harvest 2-5 days later (shorter time for more cells) But of course always let them grow at least 2 days to recover from trypsinization.

I sent some cells to a researcher who as concerned about the appearance. She sent me the attached picture. Remember hat HIT-T15 are not a single cell type, so there are beta cells, but also alpha cells, delta cells, PP cells. They tend to grow in clusters and will not spread out. You must split once a week even if the cells are not as confluent as you like. Ideally the cells are single cells when plated.

I just dicovered that my cell protocols got corrupted when a coputer virus made its way through our institution recently, so I can't send immediately, but I will put up a link as soon as I can access the files.

Hi Shabbir,

Our IT department was able to restore my files - happy day! So attached are our basic protocols for thawing, feeding, splitting, plating, freezing, and performing a basic glucose challenge experiment. They are written for a person who has never done tissue culture.

Enjoy, and good luck with your research!

Oh, also, you can thaw the cells the usual way, spinning them down and changing the media right away to get rid of the DMSO, but we usually simply place the cells into a flask and change media the next day.