Blood cells , specially RBC, WBC and Platelets plays important role in body immunity and helps to maintain homeostatic condition of the body.
For different purpose, we need to analyse the impact of different materials on blood cells. It's also not feasible to collect blood from donor or patient.
Is there any method, where we can culture blood cells in laboratory condition with ensuring 100% micro-environment along with supplementary nutrients ?
Dear Babu Mia,
You can culture lymphocytes for 3 days at 37oC degrees with 5% CO2 in different cell culture medium such as RPMI 1640 with 10% Fetal Bovine Serum (FBS) or with DMEM containing 10% FBS for screening your biomaterials. Before you culture cells, you need to separate lymphocytes by density gradient separation or magnetic beads. There are other methods, also. You maybe able to reach to book I have mentioned before, through university library, if not, please write me again to send in your e-mail. One of my students is studying on cytotoxicity experiments for biomaterials, she will correspond with you and share her protocols.
For cytotoxicity tests it is always good to start up with primary cells obtained from healthy donors. If that is not possible, depending on the type of material and its purpose of use, you can use cell lines which can be obtained from ATCC, Lonza and other providers. Methods in Molecular Biology published an issue named "Basic Cell Culture Protocols Editors: Helgason, Cheryl D., Miller, Cindy L. (Eds.) 2013 DOI: 10.1007/978-1-62703-128-8" which is very helpful.
Dear Gulderen Yanikkaya Demirel ,
Thanks for your wise reply. You are right. I always do experiment with primary cell line. Cell line dependent work is also good. But at some extent, we need to keep our cell well and sound for little bit longer time. For that longer period of time, we need to ensure microenvironment for cells. In terms of blood cells, we need to be more careful from various prospect. If there was any technique to culture them at laboratory condition, that might was supposed to be best. If we can't do that, at least , at 37'C , if we can keep them for 72 hours or more with efficient nutrient supply, that will also work. Are you acknowledged about it ?
Dear Babu Mia,
You can culture lymphocytes for 3 days at 37oC degrees with 5% CO2 in different cell culture medium such as RPMI 1640 with 10% Fetal Bovine Serum (FBS) or with DMEM containing 10% FBS for screening your biomaterials. Before you culture cells, you need to separate lymphocytes by density gradient separation or magnetic beads. There are other methods, also. You maybe able to reach to book I have mentioned before, through university library, if not, please write me again to send in your e-mail. One of my students is studying on cytotoxicity experiments for biomaterials, she will correspond with you and share her protocols.
@Gulderen Yanikkaya Demirel Thanks a lot for your kind reply. I'm using Lympholyte to separate WBC and performing my experiments. I'm mostly worried about Red blood cells , because they lyse faster.
Which bio-materials on which cells you are using to find out cytotoxic effect ? Just curiosity . ( don't share , if you are not comfortable )
We work with healthy donor lymphocytes and platelets (for plt activation effects of the biomaterials). Most of the biomaterials produced by another institute are zinc and ferrous based.
Please anyone can provide a detailed protocol for PBMC primary cell culture using RPMI media?
Mr. Nithiyanandan Saravanan ,
PBMC's are mostly mature in form and couldn't be cultured further. If you are intended to evaluate the affect of any agent or regulatory factor in lineage specific differentiation and maturation of PBMC, you need to start with Haematopoietic stem cells ( HSC ). HSC can be stimulated and guided towards the lineage specific differentiation by multiple cytokines . Downregulation or upregulation of any factor can have immense impact on the way of their differentiation. For example, Pax5 for B cells maturation.
Please feel free to knock me for further details ( if needed ) as i work on HSC to PBMC differentiation.
Reference- Cobaleda, C., Schebesta, A., Delogu, A. and Busslinger, M., 2007. Pax5: the guardian of B cell identity and function. Nature immunology, 8(5), p.463.
Best regards,
Babu M.
- Badges
- Science method