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\begin{document}

\title{Current Utilization Trend of Mast Cell Lines in In Vitro Allergy
Research: A Systematic Review}



\author[1]{Ashley Jia Wen  Yip}%
\author[2]{Yu Zhao  Lee}%
\author[2]{Audrey Siew Foong  Kow}%
\author[1]{Carisa Su-Ann Wong}%
\author[2]{Ming-Tatt  Lee}%
\author[3]{Chau Ling  Tham}%
\author[1]{Ji Wei Tan}%
\affil[1]{Monash University Malaysia School of Science}%
\affil[2]{UCSI University}%
\affil[3]{Universiti Putra Malaysia Fakulti Perubatan dan Sains Kesihatan}%


\vspace{-1em}



  
  \date{March 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Today in the modern world, allergic diseases, also described as atopic
allergies, are classified as a type of multifactorial disorder due to
the complex interplay between genetics, environment, and socioeconomic
factors that influence the disease's manifestation, severity, and one's
predisposition to allergic diseases. It is undeniable that many reported
studies have pointed out that the mast cell is one of the main key
players involved in triggering an allergic reaction. In order to improve
our current understanding of the molecular and cellular mechanisms
underlying allergy, various mast cell lines have been employed in vitro
to study the pathogenesis of allergic diseases for the past decades.
However, there is no consensus on many fundamental aspects associated
with their use, such as the effects of culture media composition and the
type of inducer used for cell degranulation. As the standardization of
research protocols and disease models is crucial, we hereby present the
outcome of a systematic review of scientific articles that used three
major in vitro mast cell lines (HMC-1, LAD2, and RBL-2H3) to study
allergy. This systematic review described the cell source, culture
conditions, inducers used for degranulation, and mediators released for
examination. We hope that the present systematic review may help to
standardize the use of in vitro mast cell lines in allergy research and
serve as a user's guide to understand the fundamental aspects of allergy
as well to develop an effective allergy therapy in the future for the
betterment of human good health and wellbeing.%
\end{abstract}%



\sloppy


\textbf{Current Utilization Trend of Mast Cell Lines in \emph{In Vitro}
Allergy Research: A Systematic Review}

\textbf{Current Utilization Trend of Mast Cell Lines in \emph{In
Vitro}Allergy Research: A Systematic Review}

Ashley Jia Wen Yip\textsuperscript{1}, Yu Zhao Lee\textsuperscript{2,3},
Audrey Siew Foong Kow\textsuperscript{4}, Carisa Su-Ann
Wong\textsuperscript{1}, Ming-Tatt Lee\textsuperscript{4,5}, Chau Ling
Tham\textsuperscript{6,7}, and Ji Wei Tan\textsuperscript{1*}

\textsuperscript{1} School of Science, Monash University Malaysia, Jalan
Lagoon Selatan, Bandar Sunway, 47500 Subang Jaya, Selangor, Malaysia.

\textsuperscript{2} Office of Postgraduate Studies, UCSI University,
56000 Cheras, Kuala Lumpur, Malaysia.

\textsuperscript{3} Faculty of Applied Sciences, UCSI University, 56000
Cheras, Kuala Lumpur, Malaysia.

\textsuperscript{4} Faculty of Pharmaceutical Sciences, UCSI University,
56000 Cheras, Kuala Lumpur, Malaysia.

\textsuperscript{5} Graduate Institute of Pharmacology, College of
Medicine, National Taiwan University, Taipei 10051, Taiwan.

\textsuperscript{6} Department of Biomedical Science, Faculty of
Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia.

\textsuperscript{7} Natural Medicine and Product Research
Laboratory~(NaturMeds), Institute of Bioscience, Universiti Putra
Malaysia, 43400 Serdang, Selangor.

*Correspondence:

Dr Tan Ji Wei

tan.jiwei@monash.edu

Running Head: Current Trend on Cellular Mast Cell Research

\textbf{Abstract}

Today in the modern world, allergic diseases, also described as atopic
allergies, are classified as a type of multifactorial disorder due to
the complex interplay between genetics, environment, and socioeconomic
factors that influence the disease's manifestation, severity, and one's
predisposition to allergic diseases. It is undeniable that many reported
studies have pointed out that the mast cell is one of the main key
players involved in triggering an allergic reaction. In order to improve
our current understanding of the molecular and cellular mechanisms
underlying allergy, various mast cell lines have been employed \emph{in
vitro} to study the pathogenesis of allergic diseases for the past
decades. However, there is no consensus on many fundamental aspects
associated with their use, such as the effects of culture media
composition and the type of inducer used for cell degranulation. As the
standardization of research protocols and disease models is crucial, we
hereby present the outcome of a systematic review of scientific articles
that used three major \emph{in vitro} mast cell lines (HMC-1, LAD2, and
RBL-2H3) to study allergy. This systematic review described the cell
source, culture conditions, inducers used for degranulation, and
mediators released for examination. We hope that the present systematic
review may help to standardize the use of \emph{in vitro} mast cell
lines in allergy research and serve as a user's guide to understand the
fundamental aspects of allergy as well to develop an effective allergy
therapy in the future for the betterment of human good health and
wellbeing.

Keywords: Allergy, Mast cells, \emph{In vitro} , HMC-1, LAD2, RBL-2H3

\textbf{1.0 Introduction}

Humans have battled with allergies for decades. The concept of allergies
was first introduced in the early twentieth century by an Austrian
scientist named von Pirquet, describing that an allergy occurs when
exogenous substances (allergens) induce a change in reactivity in an
individual's immune system, leading to hypersensitive
reactions\textsuperscript{1}. Typical allergens are found in a wide
range of environmental substances varying in their nature and source,
including food allergens, aeroallergens like pollen, mites, and dust, as
well as chemical allergens like dyes, creams and skincare products (2).
Common allergic diseases include atopic eczema (dermatitis), rhinitis,
allergic asthma, drug, and food allergies. Allergic diseases are
considered a worldwide severe health issue, and their prevalence
comprises a substantial percentage of the population. Rhinitis and food
allergies affect 10-30\% and 8\% of the population worldwide,
respectively, while skin allergies such as eczema have a lifetime
prevalence of 20\% worldwide \textsuperscript{2}. Allergy symptoms range
from mild, such as itchiness, hives, watery eyes, and a runny nose, to
life-threatening outcome, depending on the hyperreactivity of the immune
system. The lethal and exaggerated allergic reaction known as
anaphylaxis is the primary cause of death in allergic patients
\textsuperscript{3}.

Currently, the most well-known curative treatment for IgE-mediated
allergies is allergen-specific immunotherapy (AIT). This form of therapy
involves subcutaneous administration of gradually increasing quantities
of a patient's corresponding allergen until an ideal dose capable of
stimulating immune tolerance toward the allergen is achieved
\textsuperscript{4}. Immunologic improvements in patients subjected to
AIT are associated with the production of T regulatory cells that induce
the anti-inflammatory cytokine IL)-10, which causes an early decrease in
mast cells and basophil activation and the subsequent reduction of
inflammatory mediators such as histamine\textsuperscript{5}. Despite the
efficacy of AIT, the development of immune tolerance in patients is
still an evolving area. Other short-lived first-line treatments widely
used consist of inhalation of corticosteroids, \selectlanguage{greek}β-\selectlanguage{english}adrenergic agonists,
and leukotriene modifiers in allergic asthma, or the avoidance of the
food allergen and treatment with antihistamines for mild symptoms of
food allergies are available\textsuperscript{6,7}. However, these forms
of treatment merely alleviate allergy symptoms rather than target the
underlying pathology of the disorder.

The use of \emph{in vitro} mast cell models may be able to answer and
resolve some of the issues faced with current treatment. Mast cells have
been considered the main effector cells in allergic reactions, and as a
result, they have become attractive candidates in the study of
allergenicity and sensitization mechanisms. Mast cells originate from
multipotent hematopoietic stem cells that are mainly distributed in
blood vessels located at the host-environment interface, such as the
skin, airways, and gastrointestinal tract. Their localization in the
body makes them one of the first immune cells to interact with incoming
allergens \textsuperscript{3}. As described previously, mast cells play
a central role during an allergic reaction. As mast cells are replete
with the high-affinity IgE receptor Fc\selectlanguage{greek}ε\selectlanguage{english}RI, binding of allergen-specific
IgE stimulates mast cell degranulation releases prestored
pro-inflammatory mediators such as histamine, serotonin and proteases as
well as \emph{de novo} synthesis of inflammatory mediators such as
leukotriene and prostaglandins \textsuperscript{8}. This surge in the
excessive release of such mediators rapidly triggers anaphylactic
shocks. As mast cells differentiate in the peripheral tissues from
progenitor cells in the bone marrow, CD34\textsuperscript{+} myeloid
progenitor cells, derived from buffy coats, cord blood, or bone marrow,
have been used as the primary source for the generation of mast
cells\emph{in vitro} \textsuperscript{9}. However, \emph{in vitro}
research using human mast cells pose several challenges such as having
low proliferative activity and the differentiation steps occurring
physiologically in tissues are time-consuming, difficult and expensive
to recapitulate \emph{in vitro} \textsuperscript{10}\emph{.} As such,
several commercial human mast cell lines have been generated such as the
HMC-1 (human mast cell line 1), LAD2 (Laboratory of allergic diseases 2)
and LUVA (Laboratory of University of Virginia) as well as rodent mast
cell lines such as the RBL-2H3 (rat basophilic leukemia-2H3) cell line
which are routinely used as \emph{in vitro} allergy models depending on
their specific advantages and limitations \textsuperscript{11}. Although
no model has been able to fully replicate human mast cell phenotypes,
given the right culturing conditions and experimental setup, each model
may possess some benefit over the selection of others.

To our knowledge, there is relatively limited data on large-scale
tabulated data regarding the usage of mast cell line models. Hence, in
this paper, three common mast cell line models (HMC-1, LAD2, and
RBL-2H3) used in allergy-related studies are systematically reviewed
with respect to their culturing conditions, types of inducers used, and
inducing conditions. We also draw comparative tabulations and reasonings
on the type of mast cell line used in respect of the type of laboratory
conditions and experimental purposes. The review seeks to provide
researchers with details on the characteristics and mechanisms of each
mast cell model to aid in the proper selection of models for future
studies.

\textbf{2.0 Methods}

2.1 Search Strategy

Relevant articles were identified from two different databases (Web of
Science and ScienceDirect) using keywords: allergic inflammation AND
mast cell activation. All reported research studies that use \emph{in
vitro} mast cells were included in this systematic review. Filter was
applied to include research articles in English that were published from
2018-2023. This study was conducted according to the Preferred Reporting
Items for Systematic Reviews and Meta-analysis (PRISMA) 2020 guidelines.
The last search for relevant articles in all databases was performed on
15 December 2023.

2.2 Eligibility Criteria

For this systematic review, the inclusion criteria are (1) \emph{in
vitro} mast cell studies, (2) secondary cell line culture studies, and
(3) allergy-related studies. Only published research articles from
2018-2023 were included. On the other hand, the exclusion criteria are
(1) animal work that do not involve any \emph{in vitro} mast cell
studies, (2) only involve primary cell line culture studies, and (3)
full-text not accessible.

2.3 Study Appraisal and Selection

All the articles obtained from the databases using the specific keywords
were organized according to their titles, and duplicates were identified
by the same title, authors, and year of publication. The redundant
studies were removed, and the remaining articles were screened using the
pre-defined eligibility criteria. Firstly, the title and abstract of
each article were assessed by two reviewers independently. Those that
matched the eligibility criteria were then subjected to full-text
screening to further determine their relevance. Disagreements between
the two reviewers throughout the screening process were resolved by
consensus, and the reasons for excluding the articles were recorded.

\textbf{3.0 HMC-1 cell line}

The human mast cell 1 line (HMC-1) is a well-known model used in
allergic and inflammatory disease studies. It was established from a
mast cell leukemia patient, entailing a dedifferentiated, spindle-shaped
hypogranular appearance \textsuperscript{9,12}. While it is considered
an immature mast cell, the HMC-1 cell line shares a phenotype akin to
human mast cells (HuMC). The expression of mast cell-associated markers
(i.e., histamine, heparin, tryptase, and c-kit receptor) and a similar
cell surface antigen profile render the HMC-1 cell line a strong
candidate in allergy research\textsuperscript{13,14}. HMC-1 can be
further subdivided into HMC-1.1 (HMC\textsuperscript{560}) and HMC-1.2
(HMC\textsuperscript{560, 816}), depending on the location of the
mutation in the c-kit receptor. HMC-1.1 possesses a substitution of
glycine-560 to valine (V560G), while HMC-1.2 contains the V560G mutation
and another substitution of valine-816 to aspartate (D816V)
\textsuperscript{15,16}. These mutations in the c-kit receptor cause
dysregulation in c-kit receptor expression, thus resulting in the
survival of HMC-1 cell lines independent of stem cell factor (SCF) which
is essential for mast cell proliferation, chemotaxis, activation,
differentiation, and survival \textsuperscript{17,18}. This allows the
usage of HMC-1 cells to be highly favorable in an \emph{in vitro} mast
cell research as they have a higher proliferative rate than other mast
cell lines. The doubling time of the HMC-1 cell line (1-3 days) is
described to be 10-fold faster than the LAD2 cell line (10-14
days)\textsuperscript{9,19}. In addition, when compared between the two
variant sublines, HMC-1.2 exhibits a higher proliferative rate than
HMC-1.1 due to the presence of D816V mutation \textsuperscript{20}. This
mutation leads to constitutive tyrosine kinase activation of the Kit
receptor that causes a higher proliferation rate as compared to the
HMC-1.1 which does not have the same mutation point\textsuperscript{21}.

Despite sharing a phenotype akin to HuMCs, the HMC-1 cell line contains
dissimilarities when compared to mature HuMCs. HMC-1 cell line has a low
expression of mature HuMCs markers (i.e., tryptase and chymase), with
the exception of c-kit and histamine, representing immature malignantly
transformed mast cells \textsuperscript{22}. The binding of IgE to the
high-affinity IgE receptor Fc\selectlanguage{greek}ε\selectlanguage{english}RI is crucial for the activation and
degranulation of mast cells \textsuperscript{23}. As such, the lack in
surface expression of Fc\selectlanguage{greek}ε\selectlanguage{english}RI in the HMC-1 cell line requires the usage of
a physiological stimulus, such as phorbol myristate acetate (PMA),
calcium ionophore (CI), and compound 48/80 (C48/80)\textsuperscript{24}.
Additionally, Mas-Related G-protein coupled Receptor X2 (MRGPRX2) is
known to induce mast cell degranulation, contributing to pseudo-allergic
reactions caused by small molecule drugs. The HMC-1 cell line, however,
expresses lower levels of MRGPRX2 and poor degranulation activity than
the LAD2 cell line and HuMCs. It is noted that latrunculin-B can be used
to prompt MRGPRX2-mediated degranulation \textsuperscript{9}. Overall,
the shortcomings of the HMC-1 cell line may potentially limit the
insights available to mast cell activation studies. Yet, the high
proliferative rate and stable phenotype render them a feasible model for
extensive \emph{in vitro}studies.

\textbf{3.1 Cell source and culture conditions of HMC-1 cell line}

The HMC-1 cell line is often sourced from cell banks (20 of 50), with
the most provided by Dr. Joseph H. Butterfield from Mayo Clinic (6 of
50) followed by American Type Culture Collection (ATCC) (5 of 50),
Sigma-Aldrich (2 of 50), and Korean Cell Line Bank (KCLB) (2 of 50).
Other cell banks include the Cellcook Biotechnology (CB), National
Platform of experimental cell resources (NPECC), National Centre for
Cell Science (NCCS), Wu-Han University Cell Collection Center (WUCCC),
and Chinese Academy of Sciences (CAS). In addition, HMC-1 cell line from
11 out of 50 publications were gifted by other institutions, with the
most from Eiichi Morri Osaka University (4 of 50). Following that, Prof.
Jae-Young Um from KyungHee University and Prof. Jong-Sik Jin from
Jeonbuk University contributed 2 out of 50 publications each. Other
institutes include Hoseo Univeristy (Prof. Hyun-Ja Jeong), Second
Military Medical University (ZhiLiang Yu), and Sangji University.
However, the authors did not report the original source of their gifted
HMC-1 cell line. Finally, 19 of the shortlisted publications did not
specify the cell origin. The sources of HMC-1 are summarized in Figure
2.

The composition of the growth medium used for HMC-1 cell line
propagation varies among publications. Sigma-Aldrich and MERCK recommend
HMC-1 cell line to be maintained in IMDM supplemented with 10\% Fetal
Bovine Serum (FBS), 1.2mM \selectlanguage{greek}α-\selectlanguage{english}thioglycerol, and 1\selectlanguage{ngerman}× penicillin/streptomycin
in a 37°C humidified environment with 5\% CO\textsubscript{2}. In actual
protocols employed in the publications, most of the articles maintain
HMC-1 cell line in IMDM (43 of 50), followed by RPMI-1640 (5 of 50),
DMEM (1 of 50) and IMEM (1 of 50). In addition, the media was
supplemented with 10\% FBS (45 of 50), penicillin/streptomycin (44 of
50), 2mM L-glutamine (4 of 50), monothioglycerol (3 of 50),
\selectlanguage{greek}α-\selectlanguage{english}thioglycerol (2 of 50), 10\% fetal calf serum (FCS) (2 of 50),
2-mercaptoethanol (1 of 50), amphotericin B (1 of 50), and sodium
bicarbonate (1 of 50). Table 1 summarizes the culture medium and
conditions to grow HMC-1 cells.

The choice of medium and composition are crucial to provide an optimal
environment for cell growth and survival. IMDM, a highly enriched
synthetic medium, is often recommended for rapidly proliferating and
high-density cell lines. While no studies have reported the correlation
between medium composition and the growth of HMC-1 cells, evidence shows
that DMEM and RPMI-1640 media can affect the growth and differentiation
of several cell lines \textsuperscript{25,26}. The medium's excess or
lack of calcium ion (Ca\textsuperscript{2+}) and inorganic phosphate
(Pi) may attenuate the differentiation of several cell lines. The
concentration of 1.8 mM and 0.09 mM of Ca\textsuperscript{2+} and Pi,
respectively, are optimum for cell proliferation \textsuperscript{27}.
L-glutamine is supplemented to the medium to serve as an energy source
for rapidly dividing cell lines\textsuperscript{28}. The degradation of
L-glutamine to ammonia may be toxic to the cells, where 2 to 3 mM is
sufficient to reduce cell growth. Yet, such occurrence is dependent on
the cell line \textsuperscript{29}. In addition, there are contradictory
reports on supplementing media with L-glutamine on cytokine release.
Co\selectlanguage{ngerman}ëffier et al. (2001) demonstrated the reduction in pro-inflammatory
cytokines (IL-6 and IL-8) from human intestinal mucosa by glutamine via
a post-transcriptional pathway\textsuperscript{30}. Glutamine has also
decreased the expression of leukotriene C\textsubscript{4}, monocyte
chemoattractant protein (MCP), macrophage inflammatory protein (MIP)-1\selectlanguage{greek}β,
\selectlanguage{english}tumor necrosis factor alpha (TNF-\selectlanguage{greek}α\selectlanguage{english}), interleukin (IL)-15, and IL-18 in
human intestinal mast cells, and lobectomy patients
\textsuperscript{31,32}. In contrast, several publications observed an
increase in Th1 cytokines (IL-2 and IFN-\selectlanguage{greek}γ\selectlanguage{english}) in PMACI-treated intestinal
intraepithelial lymphocytes by glutamine\textsuperscript{33}. Similarly,
IL-1 and IL-10 were upregulated in glutamine-treated lobectomy patients
\textsuperscript{32}. Other supplements, such as sodium pyruvate in
IMDM, have been shown to impair cytokine production by inhibiting
inflammatory signalling pathways\textsuperscript{34}.

\textbf{3.2 Inducers and mediators release for HMC-1 cells}

As the HMC-1 cell line lacks the expression of Fc\selectlanguage{greek}ε\selectlanguage{english}RI, an external
stimulus is required to induce mast cell degranulation. The combination
of PMA and CI is widely used (25 of 50 and 28 of 50 respectively). Other
inducers include OVA, IL-33, TSLP, protein kinase activator C, RANKL,
LPS, histamine, and more. However, three of the 50 publications did not
specify the type of inducers used. The induction time implemented by
most studies falls within 5 to 8 hours (h) (23 of 50), followed by an
hour and below (20 of 50), within 13 to 24 h (16 of 50), 2 to 4 h (13 of
50) and 9 to 12 h (2 of 50). However, four publications did not indicate
the induction time. The degranulation of mast cells can be characterized
by the concentration of cytokines secreted. Many of the publications
studied the release of TNF-\selectlanguage{greek}α \selectlanguage{english}(29 of 50), IL-6 (27 of 50), IL-1\selectlanguage{greek}β \selectlanguage{english}(20 of
50), histamine (17 of 50), TSLP (14 of 50), and IL-8 (12 of 50). Table 2
summarizes our analysis of the type of inducers, induction time, and
type of mediators often studied by researchers using HMC-1 cells.

Mast cell activation depends on the cross-linking of IgE antibodies on
the Fc\selectlanguage{greek}ε\selectlanguage{english}RI and the subsequent signal transduction cascade, including
intracellular Ca\textsuperscript{2+} mobilization, influx, and protein
kinase C (PKC) activation. As such, the combination of PMACI is a strong
candidate as an inducer owing to its ability to enhance
Ca\textsuperscript{2+} influx and activation of MAP kinases
\textsuperscript{35,36}. PMA stimulates PKC activity, while CI raises
the intracellular level of Ca\textsuperscript{2+}
\textsuperscript{37,38}. Many clinical studies have shown the
upregulation of cytokines released (Th1-related cytokines) upon PMACI
stimulation, namely the IL-2, IL-6, IL-17, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and TNF-\selectlanguage{greek}α. \selectlanguage{english}However,
these studies did not affect the expression of IL-4 and IL-10
\textsuperscript{39--41}. Another point to note in future research is
that the expressions of IL-10, IL-17, IL-22, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}are higher
in IMDM than RPMI-1640 upon PMACI stimulation due to the concentration
of Ca\textsuperscript{2+}. According to Zimmermann and his colleagues
(2015), IMDM consists of 1.49 mM of Ca\textsuperscript{2+}, while
RPMI-1640 contains 0.42mM of Ca\textsuperscript{2+}. Therefore, by
increasing the Ca\textsuperscript{2+} concentration in RPMI-1640 to 1.5
mM, the expression of cytokines is comparable to IMDM. Vice versa, the
reduction of Ca\textsuperscript{2+} concentration in IMDM resulted in a
lowered cytokine expression. Thus, implying that 1.5 mM of
Ca\textsuperscript{2+} is optimal for maximal ionomycin stimulation in
cells \textsuperscript{42}.

When compared between PMACI, lectin phytohaemagglutinin (PHA), LPS,
Con-A, and pokeweed mitogen, PMACI expresses the most potent cytokine
production in a short period without significant damage to the
cells\textsuperscript{43}. On the other hand, C48/80 acts as a
''selective'' mast cell activator by stimulating the trimeric G-proteins
and activating phospholipase C and D pathways \textsuperscript{44}.
C48/80 activation can bypass Ca\textsuperscript{2+} and PKC signal
transduction, thus beneficial when PMACI cannot be
used\textsuperscript{35}.

\textbf{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
Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}α-\selectlanguage{english}subunit \textsuperscript{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
patient\textsuperscript{45}. During this routine study, researchers
discovered cultures of mast cells with functional Fc\selectlanguage{greek}ε\selectlanguage{english}RI and Fc\selectlanguage{greek}γ\selectlanguage{english}RI
receptors that continue to proliferate in a stem cell factor-containing
serum-free media. These cells resembled CD34\textsuperscript{+}-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 \selectlanguage{greek}µ\selectlanguage{english}m
diameter. Under the electron microscope, they appeared as cells with
rough surfaces and cytoplasmic projection \textsuperscript{45}. LAD
cells highly resemble mast cells as they expressed surface Fc\selectlanguage{greek}ε\selectlanguage{english}RI,
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 \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase upon Fc\selectlanguage{greek}ε\selectlanguage{english}RI aggregation. To date, LAD2
cells have been used to study mast cell proliferation, receptor
expression, mediator release/inhibition during mast cell degranulation,
and also signaling\textsuperscript{19,46}. 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 \textsuperscript{9}.

LAD2 cells degranulate well when stimulated but they lack the ability to
generate cytokines as personally experienced by R\selectlanguage{ngerman}ådinger 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 × 10\textsuperscript{6} 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 \textsuperscript{45}.

\textbf{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 \textsuperscript{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 \selectlanguage{greek}µ\selectlanguage{english}g/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 \textsuperscript{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 \emph{c-kit} activating mutation at codon 816. LAD2 cells
were grown in a humidified incubator at 37\selectlanguage{ngerman}°C with 5\%
CO\textsubscript{2}. 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 × 10\textsuperscript{5} to 2 ×
10\textsuperscript{6} cells/mL.

\textbf{4.2 Inducers and mediators release for LAD2 cells}

As LAD2 cells express the high affinity IgE receptor, they can be
sensitized with antibody to induce degranulation. Among the 75
shortlisted articles, only 24 of them reported that they sensitized LAD2
cells with the IgE antibody. Other studies directly activate the LAD2
cells to degranulate using their chosen inducer(s). Several inducers
have been used in studies involving LAD2 cells. This includes the common
C48/80 which was being used by 36 of the studies. C48/80 is a known mast
cell degranulator that has been used in countless allergy-related
studies \textsuperscript{48}. Substance P is also commonly used as an
inducer of LAD2 cells (14 of 75). Substance P is an undecapeptide found
in the human body and it is secreted from the terminals of specific
sensory nerves \textsuperscript{49} and it is a known ligand of
MRGPRX2\textsuperscript{50}. A major product of the adrenomedullin
precursor, PAMP(9-20) \textsuperscript{49} was used by three research
groups to induce LAD2 cells in this analysis. Other inducers that were
being used to activate LAD2 cells include Tween 20, LL-37,
(R)-ZINC-3573, CST-14, Thapsigargin and complement C3a as shown in Table
4. Apart from these common inducers, several studies have analyzed the
degranulation effects of antidepressants (clomipramine, paroxetine and
desipramine), contrast media (gadopentetate meglumine, iodinated
contrast media and iopamidol), p-Phenylenediamine (PPD), P17, morphine
derivatives (thebaine and pethidine) and fluoroquinolones and many
others. PPD is a component found in hair dyes implicated to induce
immediate allergy, acute dermatitis and contact
dermatitis\textsuperscript{51}. On the other hand, P17 is a short host
defense peptide from the ant \emph{Tetramonium bicarinatum}
venom\textsuperscript{52}. LAD2 cells are commonly used to study MRGPRX2
related pseudo-allergy reactions and thus they were used to identify
several novel mast cells compounds \textsuperscript{53}. Besides that,
the effects following exposure to influenza A virus were also studied
using LAD2 cells \textsuperscript{54}. As LAD2 cells are mast cells,
some researchers chose to pre-sensitize the cells first before inducing
them with the corresponding inducers. These studies used DNP-IgE as
sensitizer (18 of 75) and then followed by either DNP-HSA, DNP-BSA,
DNP-streptomycin, or streptavidin as inducers in their studies. Other
combinations such as biotinylated IgE with streptavidin and human
myeloma IgE with anti-IgE were also used by several researchers.

From the articles analyzed, histamine (36 of 75) and \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase
(62 of 75) are the two common mediators that were often evaluated. Both
mediators are pro-inflammatory mediators that play key roles as
degranulation biomarkers \textsuperscript{55}. Other mediators include
cytokines and chemokines. Thirty out of 75 articles evaluated the levels
of MCP-1, while 28 analyzed TNF-\selectlanguage{greek}α. \selectlanguage{english}Several ILs were analyzed too, and
these were IL-8 (27 of 75); IL-6 (5 of 75) and others. Several other
mediators evaluated include 5-hydroxytryptamine (5-HT), tryptase,
MIP-1\selectlanguage{greek}α, \selectlanguage{english}MIP2, prostaglandin 2 (PGD2), leukotriene B4 (LTB4),
granulocyte-macrophage colony-stimulating factor (GM-CSF) and many
others. The induction time differs based on the type of mediators
studied. From our analysis, generally, LAD2 cells were induced between
0-1 h when \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase and histamine were to be evaluated. The
duration of 30 minutes (mins) was the most common induction period while
some groups induced the cells for 40 mins, with the longest being 2
h\textsuperscript{56}. The shortest duration used to stimulate LAD2
cells to produce \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase was 15 mins by Park et al. (2019) and
histamine for 10 mins by Sun et al. (2021) \textsuperscript{57,58}. For
the evaluation of late mediators such as TNF-\selectlanguage{greek}α, \selectlanguage{english}ILs, MCP-1 and others,
longer induction time was needed, and it ranged from 6 to 8 h, with 6 h
being the most used induction time. Two other induction times were noted
which were 12 h and between 24 and 48 h. In the study involving
influenza A virus, the cytokines and chemokines were analyzed at days 1,
2 and 4 (maximum) post infection \textsuperscript{54}. In another study
using 12 h induction time, the researchers were evaluating the effects
of substance P and PAMP(9-20) on LAD2 cells and analyzed the levels of
TNF-\selectlanguage{greek}α, \selectlanguage{english}MCP-1, IL-8 and IL-31 \textsuperscript{49}. On the induction time
of 24-48 h, the study studied the effects of P17 on LAD2 cells,
analyzing MCP-1 and MIP-1\selectlanguage{greek}α \selectlanguage{english}release \textsuperscript{52}. Table 4
summarizes our analysis on the type of inducers, induction time and type
of mediators often used and studied by researchers using LAD2 cells.

\textbf{5.0 RBL-2H3 cell line}

In mast cell research, the most common animal cell line used is the
RBL-2H3 cell line\textsuperscript{59}. The RBL cells were generated from
rats that were injected with the chemical carcinogen \selectlanguage{greek}β-\selectlanguage{english}chlorethylamine
to induce basophilic leukemia \textsuperscript{60}. The RBL cells were
adapted to suspension cell culture (named RBL-1) and can specifically
bind IgE to their surface membrane \textsuperscript{61,62}.
Nevertheless, neither an IgE/antigen trigger nor chemical stimulation by
a Ca\textsuperscript{2+}ionophore were able to cause RBL-1 to release
histamine\textsuperscript{63}. A responsive subline known as RBL-2H3
that degranulated in response to an IgE trigger was successfully
isolated from subsequent cloning of RBL cells in 1981
\textsuperscript{64}. These cells were a great model for understanding
the Fc\selectlanguage{greek}ε\selectlanguage{english}RI signaling cascades as they could be cultured in huge numbers
to examine the characteristics and binding properties of IgE which will
lead to the signaling pathways involved in degranulation
\textsuperscript{65}.

RBL-2H3 cells were widely employed as a mast cells model shortly after
they were formed. Unquestionably, RBL-2H3 cells have the advantage of
being a cell line that is simple to cultivate due to its short doubling
time (18-24 h), enabling researchers to obtain a high number of
homogenous cells easily. However, their suitability and credibility were
eventually questioned \textsuperscript{66,67}. In the early years of RBL
cells establishment, a variant with impaired cromoglycate binding had
been identified in a population of RBL-2H3 which suggest that the cell
line may not be fully homogenous \textsuperscript{68}. Although research
reports frequently referred to RBL cells as mast cell line, it was in
fact derived from basophils. Additionally, the cell line was lack of
consistency as to the physiology of basophils or mast cells as well as
findings between other research groups utilizing the same cell
line\textsuperscript{66}. Mast cells and basophils are two different but
functionally related cell types that are essential in type I
hypersensitivity. Granulocyte basophils circulate, whereas mature mast
cells are only found in tissues at the interfaces with external
environment, such as the lungs, skin, and mucosal
surfaces\textsuperscript{69}. Despite their similarities, it was
suggested that the two cell types descended from distinct
lineages\textsuperscript{70,71}. Nonetheless, numerous arguments
suggested that they may share a common origin \textsuperscript{72,73}.

Nevertheless, RBL-2H3 cells have been successfully used in
investigations on IgE binding to FceRI receptors and subsequent
downstream processes \textsuperscript{66,74}. The expression of rat mast
cell protease II (RMCP-II) \textsuperscript{75} and a similar expression
of the c-kit receptor tyrosine kinase \textsuperscript{76} in RBL-2H3
cells comparative to human HMC-1 and murine P-815 mast cells were one of
the few aspects of mast cell physiology that supports the notion that
RBL-2H3 cells can model mast cells. Moreover, numerous studies have
found similarities between the exocytosis mechanisms of bone marrow mast
cells and RBL cells involving SNARE proteins, designating RBL cells as a
suitable model for research on MC exocytosis \textsuperscript{59}.

\textbf{5.1 Cell source and culture conditions of RBL-2H3 cell line}

Most of the RBL cell lines used in allergy research were the RBL-2H3
cell line (131 of 134), followed by humanized RBL cells (3 of 134). The
American Type Culture Collection (ATCC) is the most reported source (46
of 131) for the RBL-2H3 cell line. Other cell bank sources of RBL-2H3
includes China Center for Type Culture Collection (CCTCC) (20 of 131),
Japanese Collection of Research Bioresources Cell Bank (JCRB) (9 of
131), Korean Cell Line Bank (KCLB) (7 of 131), Procell Life Science \&
Technology (PLCT) (3 of 131), Bioresource Collection and Research Center
(BCRC) (2 of 131), Chinese Academy of Sciences Shanghai (CAS) (2 of
131), Binsui Biotechnology (SBB) (1 of 131), Cellcook Biotechnology (CB)
(1 of 131), Shanghai EK-Bioscience Biotechnology (SEB) (1 of 131),
National Infrastructure of Cell Line Resource (NICLR) (1 of 131), and
National Experimental Cell Resource Platform (NECRP) (1 of 131).
Additionally, cells were received as gifts from scientists from other
universities (4 of 131). However, the authors did not report the
original source of their gifted RBL cell line. Finally, the cell origin
was not specified in 33 out of 131 publications. Figure 4 summarizes the
cell line sources of RBL-2H3.

ATCC and BCRC recommends RBL-2H3 to be cultured in MEM that contains
Earle's Balanced Salt Solution, non-essential amino acids, 2 mM
L-glutamine, 1 mM sodium pyruvate, made complete with the supplement of
15\% FBS. CCTCC and JCRB recommend using the same MEM base media with
slight reduction of FBS to 10\%. KCLB, however recommends DMEM with 10\%
FBS. For institutions other than the aforementioned above, there is no
information regarding the recommended media used for the culture of
RBL-2H3.

Review of published methods revealed significant variation in the
culture media and supplements used to cultivate RBL-2H3 cells (Table 5).
However, 11 out of 131 studies did not disclose the culture conditions
used. DMEM was the most used (69 of 131), followed by MEM (44 of 131),
MEM Eagle-alpha modification (2 of 131), RPMI-1640 (3 of 131), and EMEM
(1 of 131). For serum, media were mostly supplemented with 10\% FBS (91
of 131), followed by 15\% FBS (26 of 131), 20\% FBS (1 of 131) 17\% FBS
(1 of 131) and 5\% FBS (1 of 131). Other supplements reported include
penicillin/streptomycin (102 of 131), L-glutamine/GlutaMAX (16 of 131),
Sodium pyruvate (14 of 131) and non-essential amino acid (NEAA) (7 of
131). Two study utilized distinctive medium formulation for the culture
of RBL-2H3 cells: a concoction of 70\% \selectlanguage{greek}α-\selectlanguage{english}MEM, 20\% RPMI 1640 and 10\%
FBS with 2mM L-glutamine, as well as a mix of 41.5\% MEM, 41.5\% IMDM
and 17\% FBS with 25 mM HEPES and 120 \selectlanguage{greek}μ\selectlanguage{english}g/mL gentamicin sulphate. There
is yet to be any report on the influence of various media and
supplements on RBL-2H3 cells physiology and function.

\textbf{5.2 RBL-2H3 cell sensitization, induction, and degranulation}

Following the crosslinking of their IgE-bound FceRI by multivalent
allergens, RBL-2H3 cells, like mast cells and basophils, respond with
degranulation, releasing a variety of mediators that trigger a potent
immunological allergic response\textsuperscript{77}. The cells were
demonstrated to have a bell-shaped dose-response to anti-DNP IgE as
observed in primary mast cells\textsuperscript{78,79}. A range of
non-immunological triggers such as the C48/80 and A23187 can also cause
RBL-2H3 cells to degranulate\textsuperscript{80}. Calcium ionophore
A23187 can induce mast cell degranulation by increasing calcium entry
into the cells through pore formation or as a transporting carrier
\textsuperscript{81}. RBL-2H3 cells behave similarly to basophils and
mast cells when exposed to the A23187 \textsuperscript{82,83}. A23187 at
a concentration of 5 \selectlanguage{greek}µ\selectlanguage{english}g/mL caused RBL-2H3 cells to degranulate about
50\% of available histamine, which is 1.65\selectlanguage{ngerman}× more than that of IgE
\textsuperscript{67}. On the other hand, degranulation of MCs,
especially connective tissue MCs can be induced by polybasic compounds
like C48/80. C48/80 can interact with G\textsubscript{0} and
G\textsubscript{i} and their subfamilies of G-protein coupled receptors
(GPCRs) to activate their downstream signaling of degranulation
\textsuperscript{84--86}. However, polybasic compounds were found to be
inactive on some MC subfamilies, including RBL cells which may be due to
their lack of G\textsubscript{i-3}\textsuperscript{87,88}. Nonetheless,
depending on the culture conditions, they can develop sensitivity to
C48/80. Interestingly, quercetin can cause a rise in the expression of
G\textsubscript{i-3}'s subunits, triggering RBL-2H3 response to C48/80
\textsuperscript{88}. Another study also reported that RBL-2H3 cells
change into a C48/80 active phenotype when co-cultured with 3T3
fibroblast\textsuperscript{89}. Unfortunately, they were unable to
ascertain the factors implicated with this phenomenon.

Review on the assays performed using RBL-2H3 cells (Table 6)
demonstrated that most of the studies sensitize the cells with DNP-BSA
and later induce the cells with DNP-HSA (91 of 131) and measured the
degranulation of \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase (115 of 131) and histamine (61 of
131). The induction times were mainly reported to be within 1 h (91 of
131), with most of the studies reportedly induced RBL cells for a full 1
h (29 of 131). One study used RBL-2H3 cells for cytotoxicity assay but
did not investigate any mediators released nor used any inducers.
\selectlanguage{greek}β-\selectlanguage{english}hexosaminidase is an exoglycosidase enzyme that is associated with
degranulation, and it is released together with
histamine\textsuperscript{80}. Based on our review, \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase is
the most common marker used to measure degranulation for RBL-2H3 cells.
On the other hand, histamine had a significant role in allergy and
inflammatory reactions as it mediates the interactions between
inflammatory cells\textsuperscript{90}. The pre-stored histamine within
the granules of RBL-2H3 cells were inconclusive as wide range of
histamine content had been reported per 1 \selectlanguage{ngerman}× 10\textsuperscript{5} cells:
from 20-45 ng\textsuperscript{91}, to 100 ng \textsuperscript{92} and up
to 700 ng\textsuperscript{67}. MCs had been known to produce
pro-inflammatory cytokines like IL-4, IL-13 and TNF-\selectlanguage{greek}α \selectlanguage{english}through the
induction of TLR4 and TLR2 pathways \textsuperscript{93}. RBL-2H3 has
been observed to release up to 180 pg/mL of IL-13 and 60 ng/mL of TNF-\selectlanguage{greek}α
\selectlanguage{english}\textsuperscript{94}. Our review revealed that 48 of 105 publications
had investigated and demonstrated cytokines production in RBL-2H3 cells.
However, there are studies reported conflicting findings that RBL-2H3
cells did not respond to lipopolysaccharide induction
\textsuperscript{66,67}. Those studies also demonstrated that RBL-2H3
cells did not expressed CD14 and MyD88 that is implicated in TLR4
signaling pathway. Additionally, they also reported that RBL-2H3 cells
were also unresponsive to TLR2 ligands due to the lack of TLR2 receptors
\textsuperscript{66,67}.

\textbf{5.3 Humanized RBL cells}

As discussed above, RBL cells originated from rats. It was established
that rat IgE can bind to human IgE receptor \textsuperscript{95},
however rat IgE receptor did not recognize human IgE
\textsuperscript{96}. RBL cells expressing the high affinity IgE
receptor Fc\selectlanguage{greek}ε\selectlanguage{english}RI implied that the cell line may be suitable for the
detection of allergen specific IgE and allergens\textsuperscript{59}.
Stable transfection of human Fc\selectlanguage{greek}ε\selectlanguage{english}RI \selectlanguage{greek}α \selectlanguage{english}chain in RBL cells was achieved in
the 90s with the receptor expressed mediated antigen-induced signaling
and mediator release\textsuperscript{97--100}. These humanized RBL cells
were then utilized to investigate the ability of pure peanut allergens
and other food allergens to induce degranulation
\textsuperscript{101--104}. RBL-SX38 is one of the commonly used
humanized RBL cell lines that expresses \selectlanguage{greek}α, β, \selectlanguage{english}and \selectlanguage{greek}γ \selectlanguage{english}chains of human
Fc\selectlanguage{greek}ε\selectlanguage{english}RI \textsuperscript{105}.

The addition of a reporter gene (firefly luciferase) to humanized RBL
cells paved the way for straightforward and extremely sensitive
detection of cellular activity via the IgE receptor\textsuperscript{59}.
The first and most common system was a Nuclear Factor of Activated
T-cells (NFAT)-firefly luciferase reporter that is linked to IgE
dependent signal transduction named RS-ATL8 cells \textsuperscript{106}.
The calcium influx from stimulation of the cells activates calcineurin,
a phosphatase that dephosphorylates NFAT. This resulted in the unveiling
of the nuclear localization signal in the N-terminal and leads to
nuclear translocation. NFAT then attaches to specific promoter regions
and initiates gene transcription of the luciferase reporter
gene\textsuperscript{107}. By detecting luciferase activity with
suitable substrates and a luminescence detector, activation of these
cells may be detected \textsuperscript{106}. RS-ATL8 assay is very
sensitive and well suited for high throughput format. The assay had
since been employed for the identification of food or other allergens,
as well as determining the allergenicity of vaccines
\textsuperscript{108--110}.

From our literature retrieval, we identified two articles using the
humanized RBL cells, RBL-SX38 \textsuperscript{111,112} and one article
using the reporter humanized cells, RS-ALT8 \textsuperscript{113} to
investigate the release of \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase. RBL-SX38 was obtained from
a research group in Harvard Medical School, USA while the study using
RS-ALT8 did not specify the cells' origin. Both RBL-SX38 and RS-ALT8
cells were grown in MEM supplemented with 10\% FBS, L-glutamine and
antibiotics. One study using RBL-SX38 did not specify the culture
condition. In all three studies, cells were sensitized with allergen
specific human IgE and induced by their respective allergens.

\textbf{6.0 Alternative options for \emph{in vitro} mast cell studies}

Apart from the mentioned cell lines, there are also other alternative
options for \emph{in vitro} mast cell studies in allergy albeit not as
commonly reported. One of them is the Immortalized Human Mast Cell Line
(LUVA). This cell line was first identified and characterized by Laidlaw
and her collegues in 2011 \textsuperscript{114}. The cells were grown
from CD34+-enriched mononuclear cells derived from the peripheral blood
of a donor with aspirin exacerbated respiratory
disease\textsuperscript{114}. The authors mentioned that this cell line
can be maintained without stem cell factor to survive and proliferate
without further addition of any growth factors for approximately 2
years. In addition, LUVA cells also display high levels of normally
signaling c-kit, metachromatic cytoplasmic granules, and
Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{114}. These cells will prove valuable for
functional human mast cell studies as they can be induced to degranulate
and release various allergic mediators such as \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase,
prostaglandin D2, thromboxane A2, and MIP-1\selectlanguage{greek}β.

\selectlanguage{english}Another human mast cell line that is suitable for allergy studies is the
ROSA\textsuperscript{KIT} mast cells. This cell line was first reported
in 2014 \textsuperscript{115}. Unlike LUVA cells, these cells require
stem cell factor to survive and proliferate with a doubling time of 24
h\textsuperscript{115}. ROSA\textsuperscript{KIT} cell also has
functional IgE receptors which allows it to be easily activated to
release mediators whenever Fc\selectlanguage{greek}ε\selectlanguage{english}RI crosslinking
happens\textsuperscript{115}. Apart from activation through IgE
receptors, this human mast cell line can also be induced by means of
calcium influx. A reported study showed that the
ROSA\textsuperscript{KIT} cell induction using calcium ionophore A23187
was able to release 80\% of \selectlanguage{greek}β-\selectlanguage{english}hexosaminidase as compared to using
IgE-Fc\selectlanguage{greek}ε\selectlanguage{english}RI which only releases approximately 38\% of the enzyme after 1 h
induction\textsuperscript{115}.

Apart from that, secondary mast cell lines from mouse origin such as
MC/9 and P815 cell lines are also available. The MC/9 is a cloned mast
cell line derived from the fetal liver of a (B6 \selectlanguage{ngerman}× A/J)F1
mouse\textsuperscript{116}. These cells can be sensitized to specific
antigens by incubating them with IgE having the desired antigenic
specificity, resulting in the release of soluble mediators such as
histamine and leukotrienes when induced \textsuperscript{117}. A
reported study by Jin et al. (2019) has also induced these cells using
PMA (50 nM) and A23187 (1 \selectlanguage{greek}μ\selectlanguage{english}M) for 24 h to measure the release of
histamine and LTC4 levels \textsuperscript{118}. P815 cells, on the
other hand were isolated from a mouse with mastocytoma in
1957\textsuperscript{118,119}. These cells do not express the IgE high
affinity receptor and, therefore, can only be activated independent of
the IgE pathway. However, they do express fragment crystallizable gamma
receptor II (Fc\selectlanguage{greek}γ\selectlanguage{english}RII) and could bind the Fc portion of mouse IgG
antibodies through their fragment antigen-binding (Fab) which may
recognize NK cells activating receptors leading to target cell
lysis\textsuperscript{120}. In addition, one study reported the use of
C48/80 and CI to induce degranulation in P815 cells to release allergic
mediators such as IL-4 and histamine \textsuperscript{121}.

\textbf{7.0 Conclusion}

Using secondary mast cell lines to conduct research in allergy has come
a long way since the discovery of mast cells in
1863\textsuperscript{122}. Today in modern research, the scientific
community is able to utilize different mast cell lines to study the role
of mast cell in an allergic response. This systematic review illustrated
the popular secondary mast cell lines that are used to study the
molecular and cellular mechanisms involved in functional studies in
allergy diseases, and to determine potential protective compounds for
allergic treatments (Figure 5). Therefore, some of these well-known cell
lines such as RBL-2H3, HMC-1, and LAD2 cells have been proven to be an
asset in unraveling the molecular complexity of allergy. However, every
cell line will have its own drawbacks such as absence of high affinity
IgE receptor and long doubling time. In addition, the differences in
cell source and maintenance in culture may influence the epigenetic
character of these cells which potentially lead to results variations
and inconsistency across laboratories. Thus, researchers must take into
consideration of these factors before selecting the appropriate mast
cell line for their studies.

\textbf{8.0 Availability of data and material}

Data available on request from the authors.

\textbf{9.0 Author Contributions}

YZL, ASFK, CSAW, and AJWY performed database searching, article
screening, study selection and appraisal, and data extraction and
analysis. JWT, YZL, ASFK, CSAW, and AJWY conceived the idea and wrote
the manuscript. JWT supervised the study and resolved conflicts between
reviewers (YZL, ASFK, CSAW, and AJWY). CLT and MTL provided important
information for the completion of the manuscript. All authors read and
approved the final manuscript.

\textbf{10.0 Acknowledgements}

The authors would like to thank Ms Natasha Lee Yun Zhi and Ms Chen Shuyu
for helping out in database searching, article screening, study
selection and appraisal, and data extraction and analysis for this
study.

\textbf{11.0 Funding}

This study was supported by Fundamental Research Grant Scheme (FRGS)
2018 from Ministry of Higher Education of Malaysia
(FRGS/1/2018/SKK06/MUSM/03/1) and High Impact Research Support Fund
(HIRSF) 2022 from Monash University Malaysia (STG-000170).

\textbf{12.0 Conflict of Interest}

The Authors have declared that there are no conflicts of interest.

\textbf{13.0 Consent for publication}

The manuscript has been read and approved for publication by all the
named authors.

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