Mast cells are tissue-resident cells of hematopoietic origin which contribute to adaptive and innate immune responses (Mekori et al. 2000
, Metcalfe et al. 1997
). However, it is their central role in generating and secreting the inflammatory mediators responsible for anaphylaxis and atopy that has primarily driven the research in the field of mast cell biology. Mast cells develop from CD34+
(KIT) progenitor cells which originate in the bone marrow then migrate to, and mature in their tissues of residence in response to the KIT ligand, stem cell factor (SCF) (Kirshenbaum et al. 1999
, Metcalfe et al. 1997
). Two subtypes of human mast cells have been designated based primarily on the protease contents of their granules: MCTC
, whose granules contain both tryptase and chymase, and MCT
whose granules primarily contain tryptase. The human MCTC
is considered to be more analogous to the connective tissue mast cells described in rodents, whereas the human MT
subtype is more analogous to the mucosal mast cells described in rodents (Metcalfe et al. 1997
Mature tissue-resident mast cells are principally activated via the high affinity receptor for IgE (FcεRI) following binding of antigen to antigen-specific IgE occupying these receptors (Kraft et al. 2007
), resulting in the release of inflammatory mediators as described in the “Measuring Mast Cell Mediator Release” unit. This response however can be markedly upregulated or downregulated by ligation of various other receptors expressed on mast cells. For example Kit, and various G protein coupled receptors (GPCRs) and Toll-like receptor (TLRs), when activated, enhance mast cell degranulation (Kit, GPCRs) and/or cytokine production (Kit, GPCRs, TLRs), whereas other cell surface receptors such as other GPCRs, FcγRIIb, and other ITIM-containing inhibitory receptors can down-regulate FcεRI-mediated mast cell activation degranulation and cytokine production (Gilfillan et al. 2006
The majority of studies on mast cells, to date, have been conducted in cells of rodent origin, particularly the RBL 2H3 rat mast cell line and mast cells derived from the bone marrow of wild type, transgenic and gene knockout mice. The primary reasons being the ease of obtaining large numbers of cells, ease of triggering the cells with available reagents, ease of transduction, and the ability to generate or obtain genetically modified mice allowing delineation of required signaling events. Until recently, studies on human mast cells have been limited; largely being restricted to cells derived from human tissue such as lung and skin following protease digestion and purification through cell separation gradients. The difficulty in maintaining consistency in sources of the starting tissues and the digestion and purification procedures however have the potential to compromise the viability of the mast cells isolated in this manner and to introduce significant batch to batch variability.
Studies aimed at the identification of mast cell progenitors and their requirements for subsequent differentiation, expansion, and development into mature mast cells have led to the evolution of protocols for that has allowed relatively high numbers of pure populations of human mast cells to be attained. The major sources for the progenitors obtained for these protocols have been cord blood and CD34+ progenitors obtained from peripheral blood. Peripheral blood contains a relatively low percentage of CD34+ cells (0.01–0.1%), therefore, it is necessary to enrich this population prior to growing the cells. CD34+ cells are commercially available, however, in our laboratory, we obtain the cells from normal donors, following G-CSF injection which mobilizes bone marrow progenitors. The normal donors are subjected to apheresis, and the CD34+ cells are purified by means of positive selection (all through a protocol approved by the NIAID/NIH IRB committee). We routinely obtain around 2–3 × 108 CD34+ cells per normal donor which are subsequently stored under liquid nitrogen until use.
Cord blood contains a high number of progenitor cells which makes this progenitor source favorable compared to peripheral blood. This difference in mast cell yield between cord blood and peripheral blood is, however, not related to progenitor type since the mast cell yield from peripheral blood do not increase when starting out with CD133+
cells (Holm et al. 2008
). The high yield of mast cells makes the cord blood protocol amenable for studies like microarray analysis which needs a high cell number but it has to be taken into account that the cord blood derived mast cells are different from the peripheral blood derived. They contain less histamine, express fewer FcεRI and CD203c receptors but more CD117, and when activated through FcεRI they release less histamine, prostaglandin D2
and cytokines (Andersen et al. 2008
, Iida et al. 2001
). It is therefore important to consider the mast cells generated from either cord blood or peripheral blood as two distinct types of mast cells.
As discussed in Unit 3.23, fungal, yeast, mycoplasm and bacterial contamination can present a problem, particularly as the human mast cell cultures take around 8 weeks before they are mature. Fungal, yeast, and bacterial infections are readily discernable by eye and mycoplasm contamination which is usually suspected when cells are growing slower than expected, can be detected by commercially available kits. Should contamination occur, immediately bleach and discard all contaminated flasks and scrub and clean all surfaces that have contacted these flasks with 70% ethanol.
Evidence of slower than expected growth rates or signs of cell death during the later stages of growth may also indicate problems with the growth media or batches of cytokines or SCF. It is to be expected to see a substantial amount of debris around week 3 of culture, however, due to die off of the non-mast cell committed lineages.
LAD2 cells grown for prolonged periods that display excessive clumping or slower growth may lose responsiveness to biotinylated IgE/SA crosslinking, and have reduced activation and degranulation. Clumping of cells can be minimized by maintaining cell concentrations between 0.25–0.5 × 106 cells/ml and performing hemidepletions every 3–4 days. A complete media change to spin off cell debris should be done as necessary by spinning cells at 200 × g for 5 minutes and replacing with fresh StemPro34 with rhSCF. Gentle pipetting of suspended cells with 1000 µl pipette tips will break up most cell clumps. In the event that cell degranulation or growth rates do not improve, thaw out an aliquot of frozen cells and restart LAD2 cell stocks.
For CD34+-peripheral blood derived human mast cells, we start with 1 ×107 CD34+ cells which after 8 weeks in culture yield approximately 3–10 ×107 mature human mast cells with a high degree of purity (~99%).
One hundred ml of cord blood gives around 1 × 106 CD133+ cells which proliferate into approximately 1 × 108 mast cells.
The doubling time for LAD2 cells is approximately 10–14 days and HMC1 cells, 1–2 days.
Growth and culture of primary human mast cells from 1 ×107 blood-derived CD34+ cells takes approximate 30 min to 1 hour a week, depending on the rate of growth, which may vary between different batches of CD34+ cells. The cells are mature after 7–8 weeks (with a purity >95%) at which point they can be used.
Purification of cord blood CD133+ cells takes approximately 3 hours. Culturing of the cells is similar to the peripheral blood CD34+ cells described above.
Starting up a culture of LAD2 cells take around 6 hours. The cells grow slowly initially, but should start doubling in 2–4 weeks after thawing. Cells need to be fed once a week which takes around 15–30 min depending on how many cultures you have.
Starting up a culture of HMC1 cell lines takes around 30 min. Cells expand fast and usually need to be passaged / fed every 3–5 days, which takes approximately 15–30 min depending on numbers of cultures.