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.