4.0 LAD2 cell line
Prior to the discovery of LAD cells, HMC-1 cells were the only cell
culture available to researchers that resembled human mast cells.
However, HMC cells’ usefulness is limited by two deficiencies – they
are growth factor independent and they degranulate inconsistently to
IgE-dependent signals possibly due to the variable expression of the
FcεRI α-subunit 45. Laboratory of Allergic Diseases 2
(LAD2) human mast cells were first discovered through a routine study of
cells from bone marrow aspirates of a mast cell sarcoma/leukemia patient45. During this routine study, researchers discovered
cultures of mast cells with functional FcεRI and FcγRI receptors that
continue to proliferate in a stem cell factor-containing serum-free
media. These cells resembled CD34+-derived human mast
cells and responded to human recombinant c-kit receptor ligand stem cell
factor (rhSCF) while sharing similar characteristics with LAD1 cells.
Morphologically, LAD2 cells stained with acid toluidine blue and
tryptase are oval or round nucleated cells with metachromatically
staining granules and they measured between 8 to 15 µm diameter. Under
the electron microscope, they appeared as cells with rough surfaces and
cytoplasmic projection 45. LAD cells highly resemble
mast cells as they expressed surface FcεRI, cluster of differentiation
(CD) 4, 9, 13, 22, 45, 64, 71, 103, 117, 132, C-C chemokine receptor
type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4) and CD14, 31
and 32 on a lesser degree. They can release histamine and
β-hexosaminidase upon FcεRI aggregation. To date, LAD2 cells have been
used to study mast cell proliferation, receptor expression, mediator
release/inhibition during mast cell degranulation, and also signaling19,46. In addition, these cells are commonly used in
studies involving MRGPRX2. The MRGPRX2 is expressed by mast cells and
degranulates upon binding by different ligands; and is involved in
pseudo-allergic reactions, chronic spontaneous urticaria, atopic
dermatitis and allergic asthma 9.
LAD2 cells degranulate well when stimulated but they lack the ability to
generate cytokines as personally experienced by Rådinger et al. (2010).
Apart from that, Rådinger et al. (2010) also noted the relatively slow
growth rate of LAD2 cells – doubling rate of approximately 2 weeks.
Although LAD2 cells require longer time to proliferate compared to some
tumorigenic cells which take 3 to 5 days; Kirshenbaum et al. (2003)
believed that this longer duration allowed the cells to exhibit a more
mature phenotype. Other potential drawbacks of LAD2 cells are excessive
clumping when the cells were grown for a prolonged duration and the
slower growth may hampered the cells’ responsiveness to biotinylated
IgE/streptavidin crosslinking and thus reducing activation and
degranulation. However, this could be easily overcome by maintaining the
cell concentrations between 0.25-0.5 × 106 cells/mL to
reduce cells clumping and performing hemidepletions every 3-4 days as
suggested by Rådinger et al. (2010) and to freeze down cells frequently,
then thaw and expand a new stock culture yearly 45.