IntroductionIn the past cultureware has mostly been focused on culturing standard cell lines that have historically been easy to culture. However, with the explosion of stem cell culture and the now common practice of culturing many different cell types, cultureware has needed to evolve as well. Over the years, improvements have been developed to increase the success of cultureware and to provide a more hospitable cell-growing environment, including innovations like gas-plasma treatment and biological extracellular matrices. Yet there is still opportunity to improve cultureware and address some of the most common challenges cell culture scientists face. Please join us and submit your questions about cultureware, please share your challenges and also questions you may have about the latest products in this area.
Have you thought about how one can reduce money spent in tissue culture work by reducing the amount of media used?
One observation that comes up in looking at tissue culture flasks is that not all flasks are designed equally. Flask bottoms that connect to the neck via a slanted slope actually is a non-treated area where cells do no grow, but media covers regardless. So one train of thought is to utilize a flask in culture in which the entire bottom is treated for cell culture, thus maximizing cell growth area and minimizing media wastage. Open for discussion on your thoughts or suggestions.
Great question. To more specifically answer your concern, can you please detail what are some of the challenges in growing stem cells in serum-free culture? Is attachment an issue? Is propagation or harvestation an issue?
Absolutely. This is a common problem faced by 96-well plate users referred to as Edge Effect. The uneven evaporation of media in wells within the 96-well plate, especially in the outer most wells is due to uneven heating and insulation of the plate when stacked. One way to get around this problem, which also has downstream effects resulting in variable read outs across replicates, is to use cultureware that prevents insulation and promotes even distribution of temperature. This was designed by scientists who work for the cell culture manufacturer TPP in Switzerland; they created all their flasks, dishes and plates to have a raised bottom and essentially have feet such that it creates vents for temperature to distribute evenly around the plate and most importantly under the plate. I highly recommend trying out a sample of a TPP 96-well plate for more consistent gene and protein reporting across replicates in a 96-well format. Hope this answers your question.
To follow up on my serum free question – I am having problems with attachment. Cells start out ok but then end up detaching quickly after being in culture.
I apologize for not being an expert on this topic, and without knowing the exact cell type of stem cells, I was able to look up the following 2 articles that showcase protocols for working with stem cell line expansion in serum free media:
Also, have you considered growing the stem cells on a coating to prevent detachment in a serum-free media:
I hope this points you in the right direction and hopefully we get some additional comments to aid the detachment issue.
In your blog on the dish, you mention cultureware that has ergonomic considerations. As someone who suffers from on and off carpal tunnel syndrome from pipetting, I am always interested in learning about ergonomic products for scientists. Can you give more details?
Thank you for your question. I am sorry about your carpal tunnel syndrome as this condition is really painful and affects your daily functioning in a lab. To answer your question, there are several ergonomic features incorporated within the cell culture flask, dish or plate to enable better handling and easier access to cells during culture.
TPP flasks are shorter in length to only provide you a fully treated cell growth area with zero dead space but also a better grip on the flask during handling. In the mapping out and designing TPP cultureware, the scientists made sure that hard to handle products like well plates had grip edges on the sides for better handling. Also, each plate, dish and flask, when placed in a stack, interlocks with each other to prevent toppling over during transport from the incubator to the hood and vice versa. The TPP dish has the most pronounced grip edge ring to a) prevent accidental dropping of the dish bottom from not grabbing the dish as a whole and just lifting off the top and b) to prevent contamination of the inside of the dish with the accidental exposure to the gloved hand during lid lift off. Another key product that is ergonomic is the TPP serological pipettes that are shorter in length and hence prevent the back and neck strain that results from hours of constant pipetting in the hood.
All in all, a lot of thought, design and ingenuity laid the foundation for the production the Premium TPP cultureware and I hope I was able to provide you with some examples to demonstrate that. Samples of any of these products are available upon request at http://shop.midsci.com/scategory/M50/978/Tissue_Culture/TPP_Tissue_Culture_Plastics/
Our lab has suffered from some contamination problems in our shake flasks. It seems that we are following proper protocol, but seem to be susceptible. Any suggestions on shake flask improvements that might reduce the risk of contamination?
Thank you for your question. Can i please ask a little more information on what type of shake flasks are you using? Is it flat bottomed and has a loosened cap or is it a conical bottomed Bioreactor with a screw cap that possesses a hydrophobic membrane? The latter was designed to repel media upon contact with the cap during shaking and thus prevent media accumulation and the consequent risk of contamination. Alternatively are the flasks being reused via a cleaning and autoclaving methodology or is it a sterile unit opened in the hood every time. The latter may be the most popular route for time savings and convenience but more importantly to avoid any cross contamination.
Another thing is to possibly modulate is volume of shaking and speed of shaking if vigorous media sloshing is observed during the shaking duration; however your question stated that you are following proper protocol, so I would think that this point was already looked discussed and tested.
What is the function of Ethanolamine for the growth of the CHO cells and what concentrations are generally used in the media.
Thank you for your question. I am not an expert in that field but I have found you a couple of articles that may point you in the direction requested: http://www.jbc.org/content/272/31/19133.full
Hope you find these articles helpful.
Is there a special group of plates designed for culture of stem cells. Would any of these innovations improve stem cell culture?
That is a great question. I am not 100% sure but I did find this link that you may want to look into: http://www.stemcell.com/en/Products/All-Products/96Well-Treated-Tissue-Culture-Plate.aspx
We are culturing small volumes of hybridoma cells in standard shake flasks. Are there any improved cultureware that you think would improve our process in general?
Shaking in flasks has come a long way from the ancient glass bottles to flat bottomed flasks to now the conical bottomed TubeSpin Bioreactors. The latter allows for greater gas exchange and internal cyclonic mixing to promote increased cell propagation. Take a look at the two following links and we would suggest sampling the TubeSpins to test it for yourself: Diagram: http://www.tpp.ch/page/downloads/TubeSpin/TubeSpin_vs_Erlenmeyer.jpg ; Poster: http://www.tpp.ch/page/downloads/TubeSpin/2012_ECI_Poster_Monteil.pdf