Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application

Titelangaben

Helm, Moritz ; Riedl, Simon ; Gollner, Katrin ; Gollner, Ulrich ; Jérôme, Valérie ; Freitag, Ruth:
Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application.
In: Journal of Chromatography B. Bd. 1179 (2021) . - 122853.
ISSN 1873-376X
DOI: https://doi.org/10.1016/j.jchromb.2021.122853

Angaben zu Projekten

Abstract

B lymphocytes (‘B cells’) are components of the human immune system with obvious potential for medical and
biotechnological applications. Here, we discuss the isolation of primary human B cells from both juvenile and
adult tonsillar material using a two-step procedure based on gradient centrifugation followed by separation on a
nylon wool column as alternative to the current gold standard, i.e., negative immunosorting from buffy coats by
antibody-coated magnetic beads. We show that the nylon wool separation is a low-cost method well suited to the
isolation of large amounts of primary B cells reaching purities ≥ 80%. More importantly, this method allows the
preservation of all B cell subsets, while MACS sorting seems to be biased against a certain B cell subtype, namely
the CD27 + B cells. Importantly, compared to blood, the excellent recovery yield during purification of tonsillar B
cells provides high number of cells, hence increases the number of subsequent experiments feasible with identi-
cal cell material, consequently improving comparability of results. The cultivability of the isolated B cells was
demonstrated using pokeweed mitogen (PWM) as a stimulatory substance. Our results showed for the first time
that the proliferative response of tonsillar B cells to mitogens decline with the age of the donor. Furthermore, we
observed that PWM treatment stimulates the proliferation of a dedicated subpopulation and induces some termi-
nal differentiation with ASCs signatures. Taken together this indicates that the proposed isolation procedure pre-
serves the proliferative capability as well as the differentiation capacity of the B cells.