I am going to culture cancer stem cells from HCC cell line Huh7. But I do not know how to set out to do. What kinds of medium should I use? Does it need any growth factor such EGF? Would anyone like to teach me? Thanks.
Hello Yongyu Shi
It is difficult to maintain Cancer Stem Cells (CSCs) efficiently in vitro because they form a very small proportion of cells, and are prone to differentiation in culture, and require a specific microenvironment for stemness.
First and foremost, you need to identify and isolate CSCs. There are basically four main methodologies that depend either on the high expression of cell surface markers or on the functional aspects of CSCs. The most commonly used method of CSC isolation is based on specific cell surface biomarkers or biomarker combinations, which is also a priority in cancer research. The other three commonly used methods, i.e., side population (SP) cell isolation with Hoechst 33342, ALDEFLUOR assay, and mammosphere formation, are mainly based on CSC intrinsic properties. These methods have been widely applied to isolate CSCs from cancer cell lines.
Several studies have recorded CD133 expression on the surface of CSC cells in liver cancer. According to a 2006 study, the CD133 + population in Huh-7 cells has a strong ability to proliferate in invitro culture and the ability to form tumors when injected into NOD/SCID mice. See reference attached below.
Article Suetsugu A, Nagaki M, Aoki H, Motohashi T, Kunisada T, Moriw...
You may make use of flow cytometry for the analysis of CSCs. It allows the simultaneous analysis of different cellular features with high performance and reliability. Moreover, it enables the separation of living cells on the basis of marker expression or functional properties by fluorescence-activated cell sorting. A major advantage of this technique is its ability to isolate rare cells, which is a prerequisite of identifying small cell populations within the tumor bulk. Also, a special focus is set on the side population analysis and the analysis of aldehyde dehydrogenase (ALDH) activity, which are markers that provide information about stem cell-specific metabolic functions and can be used for their identification.
In order to maintain the CSC population, you could use various commercially available media that have been developed for human ES cells and iPS cells. These media consist of basal medium and additional factors, such as basic FGF, insulin, and transferrin, and also contain other factors that are essential for cell survival and proliferation under serum‐free conditions. I would recommend the use of mTeSR1 medium as it would help to improve the viability and maintenance of CSCs for a prolong time in culture. Please refer to the link below for more details on the product.
https://cdn.stemcell.com/media/files/pis/29917-PIS_2_0_0.pdf
Hope this information may help you in your experiment!
Regards
Malcolm
Hello, Malcolm,
Thank you so much for your answer. It really helps me a lot.
Malcolm Nobre
First you need to confirm the type of cancer cells in hematopietiic stem cells; lymphocyte or myelocyte according to the type of cell you can choose the suitable cell culture; if lymphocyte also you can confirm B cell type or T cells type after that you know which growth factor or suppress factor
- Badges
- Science topic