4.1 Cell source and culture conditions of LAD2 cell line
LAD2 cells are often sourced from the Laboratory of Allergic Diseases, National Institutes of Health (NIH) in Bethesda, United States of America (USA) where the cells were first discovered and successfully cultured. Of the 75 papers analysed, 55 of the research groups obtained their LAD2 cells from NIH, specifically the laboratory of Drs Arnold Kirshenbaum and Dean Metcalfe 47. Some were obtained from Otwo Biotech company (1 of 75), Dr Yangyang Yu of Shenzhen University (1 of 75), Dr Michael of Colombia University and Professor Renshan Sun of Third Military Medical University (1 of 75). However, the authors did not report the original source of their gifted LAD2 cell line. Finally, 17 research groups did not specify the origins of their LAD2 cells. Figure 3 summarizes the most common sources to obtain LAD2 cells.
StemPro-34 is the culture medium used to culture LAD2 cells as observed in most of the 75 papers analysed (74 of 75). There is only one publication that reported the use of IMDM with supplementation of 10% FCS to culture the LAD2 cells. StemPro-34 is a specifically formulated serum-free medium used to support the growth of human hematopoietic cells. Supplements are often added in the StemPro-34 culture medium for LAD2 cells. The medium is usually supplemented with StemPro-34 nutrient supplement which include pre-mixed penicillin/streptomycin, L-glutamine, and human stem cell factor. Apart from that, Zou et al. (2022) added 50 ng/mL of IL-6 into their StemPro-34 medium. Cytokines and growth factors are added into the medium to further support the growth of the progenitor cells. The use of StemPro-34 is in accordance with the medium used when LAD2 cells were first cultured in the laboratory of Drs Kirshenbaum, Akin and Metcalfe. Similarly, the medium was supplemented with 2 mM L-glutamine, 100 IU/mL penicillin, 50 µg/mL streptomycin, 100 ng/mL rhSCF. The addition of 100 ng/mL of rhIL-6 and 30 ng/mL rhIL-3 for the first week were optional 45. Kirshenbaum et al. (2003) also noted that even though LAD2 cells could survive in medium without SCF, the numbers doubled in approximately 3 weeks with 100 ng/mL SCF, and the addition of rhIL-3, rhIL-5 or rhIL-6 did not influence the cell numbers. SCF is needed for the growth of LAD2 cells as they do not possess the c-kit activating mutation at codon 816. LAD2 cells were grown in a humidified incubator at 37°C with 5% CO2. Table 3 summarized the culture medium used to grow LAD2 cells. As suggested by Rådinger et al. (2010), hemidepletions of the medium were performed by most researchers when growing the LAD2 cells at the frequency of once a week. The cell density was generally maintained between 1 × 105 to 2 × 106 cells/mL.