Trouble with rat heart H9C2 cells

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maddensd
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Trouble with rat heart H9C2 cells

I am currently maintaining the rat heart cell-line H9C2. I am using DMEM supplemented with 2mM Glutamine, 10% FCS and Pen/Strep. I find that they grow very slowly. I also find alot of debris floating around a day after splitting them or ~three days after changing the media. I don't think that it's contamination because a microbe would have fully blossomed by 3 days. Are my doing something wrong? Does anyone else have the same problems with them?

SanDiablo
SanDiablo's picture
The following is from the

The following is from the ATCC website. Cellular debris after splits is likely the result of over trypsinization or excessive agitation during dissassociation. Try being VERY gentle...nurture your cells and speak kindly to them...

It looks like increaseing your glutamine concentration may help with the growth rate, but muscle cells do grow slowly. Also check the pH of the glutamine to ensure its availibilty. Refresh the media frequently to keep them well fed, and take the warning about cells becoming too confluent seriously, as they won't differentiate. If your culture has ever become too confluent, thaw out an earlier aliquot and start over.

If you are still having trouble, check out the orignal references and try to speak with one of the researchers that created the cell line.

Best of luck, and be sure to let the board know if this information solves your problems! We really worry about you...

San Diablo

From www.atcc.org

Comments: H9c2(2-1) is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue by B. Kimes and B. Brandt and exhibits many of the properties of skeletal muscle.

Myoblastic cells in this line will fuse to form multinucleated myotubes and respond to acetylcholine stimulation.
Fusion occurs faster if the serum concentration in the medium is reduced to one percent.

Propagation: ATCC complete growth medium: Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 90%; fetal bovine serum, 10%
Temperature: 37.0C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Subculturing: Protocol: The myoblastic population will become depleted rapidly if the cultures are allowed to become confluent.
To prevent loss of myoblastic cells, cultures should be subcultured before they become confluent, and the line should be recloned periodically with selection for myoblastic cells.

Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.

Subcultivation ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Medium renewal: Every 2 to 3 days

Preservation: Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase

References: 1062: Kimes BW , Brandt BL . Properties of a clonal muscle cell line from rat heart. Exp. Cell Res. 98: 367-381, 1976. PubMed: 943302
32970: Levy AP , et al. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J. Biol. Chem. 271: 2746-2753, 1996. PubMed: 8576250

pikayou
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I'm having the exact same

I'm having the exact same problem with my H9c2's (floating debris, slow growth, etc.) Did you ever come up with a solution or possible hypotheses?

maddensd
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I got the cells from another

I got the cells from another member of our department and she was having the same problems. I got a reply from another forum from a guy who wasn't having any problems - so I reckoned that something had gone wrong with our cells from the beginning - they constantly advise against letting the cells reach confluency - I was afraid that maybe it had happened in our department previously and that we were growing cellls that had changed. I ordered fresh cells in and broke them out yesterday - I'll post a reply next week to see how they get on.

I didn't look at glutamine pH because I use stocks that everyone uses and there hasn't been trouble with anyone else's cells. I used EDTA alone to lift cells in case the trypsin was too strong and it didn't prevent the debris problem. I never contacted the people who created the cell-line - I might try that if these cells don't work out.

SOUND
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Has anyone induced beating on

Has anyone induced beating on H9c2 cell lines?

jaqueska
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SOUND wrote:Has anyone

SOUND wrote:

Has anyone induced beating on H9c2 cell lines?

I am also trying to get them to beat... no success yet but my department head has accomplished it.

jaqueska
jaqueska's picture
How do you know when the H9c2

How do you know when the H9c2 are fully differentiated and are ready for experimentation? Do you let them grow to confluence and then pass them or do you just reduce the serum to 1%?? I am very new to this cell line and am having problems with reproducibility.

SOUND
SOUND's picture
jaqueska wrote:SOUND wrote

jaqueska wrote:

SOUND wrote:
Has anyone induced beating on H9c2 cell lines?

I am also trying to get them to beat... no success yet but my department head has accomplished it.

 
Hi....could you give the paper reference of how they induced the h9c2 cells to beat?
Also does anyone know how to improve the cardiomyocyte proliferation using the h9c2?I dont want to do any transfection is there some growth factor?
 

SOUND
SOUND's picture
jaqueska wrote:How do you

jaqueska wrote:

How do you know when the H9c2 are fully differentiated and are ready for experimentation? Do you let them grow to confluence and then pass them or do you just reduce the serum to 1%?? I am very new to this cell line and am having problems with reproducibility.

well i  dont let them grow to full confluence.generally i split them at 70% confluence and plate them for experimentation.I do imaging so i let them for a few days in well plates before i do my imaging on them.
i actually use 10% FBS for the cells ....with DMEM.
 
 

weerasak
weerasak's picture
hi

hi
I have no answer but i have a question. I will get H9C2 and i want to know how to freeze, thaw and subculture them. you please tell me clearly. thank you.

sandeepnccs
sandeepnccs's picture
I have been using H9c2 cells

I have been using H9c2 cells for last 5 years now.
 
Please use the following recommended media  DMEM with 4mM Glutamine and 1.5g/L NaHCO3 with 10% FCS and Pen/Strep or Gentamycin.   They will be 70% confluent on the third day.  Split them at 70% confluency, do not let them grow more than 70 - 80% confluent.
 
I hope this works for you.

myocyte
myocyte's picture
HI ...

HI ...
i ve started using H9c2 cell line. can somebody please guide me ..that how can i induce hypertrophy in it..?? & what can be other cell lines that one can use to study hypertrophy...?

abd265
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Hello, Im trying to culture

Hello, Im trying to culture the same cell line (H9C2 rat heart cells). I'm using DMEM high glucose medium (4nm L-lactate) and I'm having the same problem: cells are too few, small with a round shape.  A guy from our lab told me that I have to change my culture flasks ( i'm using T30 flasks) because , as he said, they are too large for my cells that are few, But I dont think this is the reason.
I'll really appreciate any answers from you that can help me with my work, thank you a lot
Abdelkader !!

Joshua Woods
Joshua Woods's picture
Same Problem passing

I'm having the same problem with the debris appearing after passing. Have you discovered a solution?

Gökçe Kaynak Bayrak
Gökçe Kaynak Bayrak's picture
detaching H9C2

I'm culturing H9C2 cells too. I m using DMEM high glucose with %10 FBS (4nm L-lactate). After trypsin/EDTA treatment cells start to clamp at 37C and never dissociate again. Also after seeding and spreading to culture plate they detaching only with PBS washing. How can ı solve the problem ?