Hi Cansu,

First, you can isolate B cells from PBMC by staining with anti-CD19 Ab and sorting by FACS or magnetic beads. Thus you can get  B cells with high purity to do the following experiment.

Secondly, you can try to express CD40L on the surface of cells as feeder layer for B cell activation. Because the natural property of CD40L is trimeric form. The activated efficiency of soluble CD40L is lower than CD40L-expressing cells. But 100 ng/ml soluble CD40L is sufficient for activating B cells in proper cell densitiy, the activated efficiency maybe elevate after excluding other PBMC cells.

Hope these can help you.

Good luck.

Weber

Thanks Weber. I am using megaCD40L, in which two trimeric CD40L are linked, so supposed to have high activity. Starting with purified cells would definitely increase my purity but would it help with viability? My main problem is that most cells are dying in culture. 

Hi Cansu,

My concern is that:

(1) There is only 5~10% of B cells in PBMC. Maybe the megaCD40L could not efficiently stimulated your B cells and leading to cell death.

(2) Other cells may consume the nutrition of B cells in culture media.

Best,

Weber 

what you get is quite normal. This method is made to temporarily increase growth of B cells isolated from PBLs.

Having 10% of living cells after 20 days of culture is a pretty good results.

However to maintain B cell clones, this is impossible. You have to go once a immortalization step, especially with EBV, which is going to stabilize culture, and proliferating B cells

In this case the use of IL4 is not necessary, it also tends to create a selection in the B cell populations and especially of activating macrophages that will be happy to devour and B cells all that is in the culture wells.

In reality, if you want to study the complete repertoire of a patient, or if you want to clone an antibody of interest, it is a bit more complicated than that.

Three steps are required:

1- must activate the B cell, and for this the CD40 system (Banchereau / Rousset) is the most compelling, and MAB 89 is superior to the different forms of CD154. but this is not specific B cells

2- must stimulate the cells; to increase the secretion of antibodies and make premissives B cells to EBV. In this case the MAB107B5 is a real plus.

3- requires a EBV virus preparation that is of high quality; the B cell growth that will ensue becomes exponential, and requires a rich culture medium without adding any cytokine.

For all this you will find many manufacturers that sell this type of reagents. it depends primarily on the target you want to achieve ..

We used the Human B Cell Culture and Expansion Kit from Humimmu.

https://humimmu.com/human-b-cell-culture-and-expansion-kit.html

It works well. I think they used irritated 3T3-CD40L cell as feeder cell.

The protein of CD40L is not good for B cell culture.