A Comprehensive Review of Current In Vitro Models for PF
1. Alveolar Epithelial Cells (AECs)
AECs are classified into two main types: type I AECs (AEC1s) and type II
AECs (AEC2s). AEC2s play a crucial role as the initial responders to
lung injury and perform essential functions such as maintaining the
alveolar environment, transitioning into AEC1s during lung injury,
releasing various pro-fibrotic and chemotactic factors, and are closely
associated with the occurrence and progression of PF. In contrast, AEC1s
are large, flat cells with limited proliferative capacity. They are
challenging to harvest and maintain in primary cultures, with AEC2s
replenishing them after injury(7). Presently, there are two main types
of AEC2 cells used to construct in vitro models of PF mainly include
primary and replacement cell lines. Primary cells refer to human or
mouse-derived primary AECs, while alternative cell lines include: (1)
Human lung cancer AEC line (A549 cell line); (2) SV40 large T transgenic
mouse-derived AEC line (MLE-12 cell line); and (3) Rat-derived type II
AEC line (RLE-6TN cell line).
1.1 Primary AECs
The isolation and cultivation of primary AECs are crucial for current
scientific experiments, providing essential insights into the cellular
and molecular understanding of PF pathogenesis. Mechanisms such as
cellular senescence, cell death, and endoplasmic reticulum stress play
pivotal roles in AEC2 depletion and impaired self-renewal, hindering the
regenerative capacity of damaged lung tissue.
Numerous pathogenesis studies of PF have utilized primary AECs to
construct pathology models. For example, Lv X et al. investigated the
regenerative capacity of lung alveoli by examining AEC2 senescence
induced by a single dose of bleomycin (BLM) and multiple doses of BLM in
a PF model(8). Liu Y et al. studied silica-treated THP-1-induced
macrophages and AEC2s, discovering that ferroptosis specifically occurs
in AEC2s rather than macrophages or fibroblasts during PF(9). To confirm
that IL-17 drives pulmonary endoplasmic reticulum stress and apoptosis,
ultimately influencing fibrosis, Cipolla E et al. used RNA isolated from
normal human AECs exposed to IL-17A for 24 hours(10).
However, the application of primary cells is limited due to unreliable
supply, challenges in isolation and in vitro culture, and the loss of
unique phenotypes over time, which hinders in vitro study.
1.2 AEC Lines
To overcome the limitations of primary cells, cell lines are often used
as substitutes for primary cells. These cell lines are typically derived
from cancer tissues or obtained by inducing immortalization through
retroviral transduction or transfection of primary cells. Compared to
primary cells, cell lines are easier to culture with the advantages of
higher proliferation rates and longer lifespans while maintaining their
original phenotypes during cultivation. Currently, cell lines are
commonly used in research to study mechanisms such as
epithelial-mesenchymal transition (EMT), cell senescence, cell
communication, and post-translational protein modifications.
The A549 cell line is a genetically mutated human non-small cell lung
cancer cell line. Though A549 cells exhibit some characteristics of
AEC2s, their structural and barrier properties, as well as cell
phenotypes, significantly differ from those of AEC2s, and they do not
express pulmonary surfactant proteins. A549 cells have been extensively
utilized as an in vitro model for lung epithelial cell injury and EMT
progression. For example, Lin C et al. conducted in vitro experiments
by inducing A549 cells to undergo EMT through TGF-β1 stimulation,
demonstrating that Valproic acid (VPA) inhibits EMT in AECs in a time-
and dose-dependent manner(11).
The MLE-12 cell line is derived from SV40 large T transgenic mice and
exhibits typical characteristics of cancer cells. MLE-12 cells can
express surfactant proteins B and C (SP-B, SP-C), similar to AEC2s.
However, like A549 cells, MLE-12 cells significantly differ from AEC2s
in terms of structure, barrier properties, and cell phenotypes. Various
studies have utilized MLE-12 cells in different mechanistic studies.
Phosphorylation, the most widespread form of post-translational protein
modification, was investigated by Pedroza M et al., who demonstrated
that STAT-3 phosphorylation is involved in TGF-β and IL-6-induced injury
and fibrosis in AECs and lung fibroblasts using immortalized AEC2s
(MLE-12 cells)(12).
The RLE-6TN cell line is isolated from 56-day-old male F344 rats through
airway perfusion using a protease solution to separate type II AECs.
These cells do not express SV40-T antigen, indicating their spontaneous
immortalization. RLE-6TN cells exhibit characteristics similar to AEC2s
and express several chemokines comparable to primary cultures of type II
alveolar cells. Furthermore, they do not form tumors in nude mice. Zhou
T et al. found that melatonin prevents cellular focal death through
nrf2-triggered ROS downregulation by exposing RLE-6TN cells to different
doses of Lipopolysaccharides (LPS) and Adenosine triphosphate (ATP)
while incubating the injury-inducing constructs in a PF pathology
model(13).
However, cell lines used as substitute models for primary cells may
express phenotypes inconsistent with their main counterparts. Therefore,
when using cell lines in medical research to simulate primary AT2 cells,
researchers should carefully consider the limitations associated with
these cell lines.
2. Bronchial Epithelial Cells
Bronchial epithelial cells serve as both physical barriers against
external damage and active participants in maintaining airway structure
and promoting lung tissue repair. Growing evidence suggests that
abnormal responses of airway epithelial cells, including AEC2 cells, are
involved in PF(14). In PF, there are also cells expressing markers of
proximal airway epithelial cells found in the distal lung, such as
goblet cells, basal cells, and ciliated cells(15). Through the release
of cytokines and growth factors, bronchial epithelial cells induce cell
differentiation, alter matrix deposition, chemotaxis, and activation,
thereby enhancing lung host defense mechanisms(16). Currently, bronchial
epithelial cell models, including primary cells and alternative cell
lines, are commonly used to construct pathological models of PF. Primary
cells consist of human primary bronchial epithelial cells (HBECs) and
mouse primary bronchial epithelial cells (MBECs). Alternative cell lines
include the human bronchial epithelial-like cell line (16HBE) and the
human bronchial epithelial cell line (BEAS-2B).
2.1 Primary Bronchial Epithelial Cells
Primary bronchial epithelial cells exhibit the most natural
characteristics of epithelial cells. Isolating and culturing these cells
from lung tissue better preserves their morphology, functional features,
and expression of lung epithelial lineage characteristics. Additionally,
primary bronchial epithelial cells retain their potential to
proliferate, migrate, and differentiate into various cell types, making
them valuable models for studying the mechanisms of lung fibrosis and
drug development(17). These cells are often utilized to investigate
mechanisms such as EMT, autophagy, cellular senescence, and apoptosis in
the pathogenesis of PF.
Interestingly, Asghar S et al. demonstrated that small extracellular
vesicles (EVs) from idiopathic PF (IPF)-injured distal human bronchial
epithelial cells (DHBEs) induce senescence in normal HBECs, potentially
creating a feed-forward loop leading to epithelial cell senescence(18).
Studies have reported that after apoptosis of AECs, bronchial epithelial
cells undergo abnormal re-epithelialization and exhibit resistance to
pro-apoptotic stimuli, contributing to the pathological activation of
epithelial-stromal interactions. Minagawa S et al. showed that effective
degradation of p21 protein through the proteasome pathway inhibits
TGF-β-induced cellular senescence in HBECs, thereby alleviating PF(19).
However, isolating and culturing primary cells is relatively costly, and
they share common disadvantages associated with primary cells, such as
strict culture environment requirements and experimental operations.
2.2 Bronchial Epithelial Cell Lines
Due to the limited feasibility of culturing primary bronchial epithelial
cells, which lose vitality after passaging, researchers often employ
alternative cell lines such as the human bronchial epithelial cell lines
16HBE and BEAS-2B. Compared to primary cell models, cell lines offer
advantages such as being of human origin, possessing physiological
barrier characteristics, and ease of culture. Human bronchial epithelial
cell lines are widely used to study the pathogenesis of PF, including
EMT, post-translational protein modification, and mitochondrial
autophagy.
The 16HBE cell line is a transformed human airway epithelial cell line
that exhibits many characteristics of differentiated bronchial
epithelial cells, including differentiated morphology and normal
function. These cells can uptake silica particles without structural
changes and express proteins typically produced by epithelial cells,
such as laminin and type IV collagen. Bodo M et al. found that when
silica particles enter the epithelium, they alter the phenotype of
bronchial cells. Using radiolabeled precursors, they studied protein
synthesis, collagen, and fibronectin in 16HBE cells maintained in vitro,
providing evidence that bronchial epithelial cells directly participate
in the pathogenesis of lung fibrosis(20).
The BEAS-2B cell line is a continuously growing cell line derived from
pathological sections of normal human bronchial epithelium, which was
infected with adenovirus 12-SV40 hybrid virus to establish an
immortalized cell line resembling respiratory lung epithelial cells.
BEAS-2B cells retain the capacity to differentiate into squamous cells
in response to serum, exhibiting traits of epithelial cells and terminal
differentiation. For example, Li X et al. investigated the ability of
mitochondrial autophagy in lung epithelial cells to reduce mitochondrial
reactive oxygen species (ROS) accumulation and protect bronchial
epithelial cells from bleomycin-induced cell death using BEAS-2B
cells(21).
Cell lines are continuously growing and differentiating cell
populations, and due to their genetic modifications, they possess the
characteristic of immortality, resulting in their potential for
unlimited growth. However, with prolonged passaging, cell lines may
undergo changes in genotype and phenotype, potentially losing the
characteristics of primary cells.
3. Macrophages
AMs are crucial innate and adaptive immune cells that originate from
progenitor cells in the bone marrow and can either circulate in
peripheral blood or migrate to different tissues. There are three major
macrophage populations: tissue-resident alveolar macrophages (TR-AMs),
monocyte-derived alveolar macrophages (Mo-AMs), and interstitial
macrophages (IMs)(22). Understanding their role in PF is of great
interest, and researchers commonly utilize primary macrophages and
alternative cell lines to study macrophage behavior and function(23).
Primary macrophages are often derived from AMs and mouse bone
marrow-derived macrophages (BMDMs), while alternative cell lines
include: (1) Human peripheral blood monocyte cells (THP-1 cell line);
(2) Mouse alveolar macrophages (MH-S cell line); and (3) Mouse monocyte
macrophage leukemia cells (RAW264.7 cell line).
3.1 Primary Macrophages
Recent research has highlighted the importance of primary macrophages in
studying PF. These cells closely resemble macrophages found in the body
and have been widely recognized and accepted by researchers. Using
primary macrophages avoids variations that may occur with genetically
modified cell lines, providing more stable and reliable experimental
results.
Notably, there are two distinct subsets of AMs in IPF: Mo-AMs and
TR-AMs. Mo-AMs produce pulmonary surfactant protein E (ApoE), which is
one of the major characteristic markers distinguishing Mo-AMs from
TR-AMs. Cui H et al. found by using Mo-AMs and TR-AMs as in vitro models
that ApoE directly binds to type I collagen and mediates its
phagocytosis leading to the alleviation of PF(24). BMDMs are terminally
differentiated cells that do not proliferate and cannot be passaged;
they can only be used for primary culture, and their survival time is
relatively short. However, these advantages also become drawbacks when
using BMDMs. The extraction process of primary cells is relatively
cumbersome and operationally challenging. Freshly isolated cells are
required for each experiment, which can be quite troublesome, and BMDMs
need to be induced for seven days before they can be used. Boutanquoi PM
et al. isolated BMDMs from trim33-floating mice and induced an in vitro
model with TGF-β1 or BLM. Liang Q et al. studied how macrophages
converted mechanical signals into IL-1β production by conducting
experiments on primary AMs and differentiated BMDMs(25, 26).
Although monocyte cell lines have significant advantages in terms of
easy accessibility compared to primary macrophages, their differentiated
state means that the conclusions drawn from these experiments regarding
the behavior of differentiated tissue macrophages may not always be
accurate. Therefore, the cytokine response induced by monocyte cells
often varies depending on the stimulation and the type of differentiated
macrophage used(27). Primary macrophages need to be collected from
specific tissues through blood donation or invasive procedures such as
bronchoscopy or tissue biopsy, which means that the number of primary
macrophages is limited and not easily expanded in vitro.
3.2 Macrophage Cell Lines
Macrophage cell lines are frequently utilized as alternative models in
scientific research to simulate macrophage functions through various
degrees of monocyte cell line differentiation. These cell lines, such as
monocyte-derived M1 and M2 macrophages, as well as foam cells, are
commonly employed in inflammation studies. Additionally, researchers
often utilize macrophage cell lines to investigate mechanisms related to
inflammation, cellular autophagy, and apoptosis in PF.
The THP-1 cell line, derived from the peripheral blood of an 83-year-old
male acute monocytic leukemia patient, exhibits characteristics similar
to primary human monocytes regarding morphology, function, and
differentiation markers. Unlike peripheral blood monocytes (PBMCs),
THP-1 has a more consistent genetic background and is easier to culture,
minimizing issues arising from PBMC variability and enhancing
experimental repeatability. Consequently, THP-1 is widely used in
various laboratories for studying immunity and inflammation. Two classic
inflammation models are commonly employed for induction and
differentiation experiments: (1) M1-type macrophage induction achieved
by lipopolysaccharide (LPS) and IFN-γ, and (2) M2-type macrophage
induction simulated by IL-4, IL-13, and macrophage colony-stimulating
factor (M-CSF) to mimic late-stage tissue repair after inflammation(28).
She Y et al. have used phorbol 12-myristate 13-acetate (PMA) to
differentiate THP-1 cells into macrophages, examining TGF-β expression
levels. Furthermore, PMA has been used to induce differentiation of
THP-1 cells into M0 macrophages, which were later polarized into M1 or
M2 phenotypes(29).
The MH-S cell line, derived from mouse alveolar macrophages transformed
by SV40, retains numerous characteristics of alveolar macrophages,
including their typical morphology and adhesive, phagocytic,
esterase-positive, and peroxidase-negative properties. Macrophage
autophagy, leading to mitochondrial autophagy, can reduce excessive ROS
release, subsequently decreasing the secretion of inflammatory factors
and ultimately alleviating CS particle-induced PF. For instance, Du S et
al. utilized cigarette smoke (CS) to induce cellular autophagy in MH-S
cells, constructing a model for PF(30). Similarly, Qian Q et al. exposed
MH-S cells to CS to establish an in vitro silicosis cell model, aiming
to determine the significance of macrophage autophagy in silicosis
development(31).
The RAW264.7 cell line was established by W.C. Raschke at the Salk
Institute in California using ascites from male BAB/14 mice induced with
A-MuLV (Abelson murine leukemia virus) through intraperitoneal
injection. This cell line serves as one of the most commonly used
inflammation cell models. RAW264.7 cells exhibit a strong phagocytic
capacity and, upon antigen phagocytosis, release chemotactic factors
that promote differentiation, extend pseudopods, and enhance crawling
ability. However, during cell culture, excessive antigen phagocytosis,
poor culture conditions, or sparse cell distribution after passage may
cause cells to exhibit spindle or elongated shapes, making digestion
more challenging. Wang Y et al. studied various in vitro cell models,
including the LPS/IL-4-induced macrophage inflammation model and the
TGF-1-induced fibroblast activation model(32). Moreover, Zhang Y et al.
induced RAW264.7 polarization into the M2 phenotype by overexpressing
TREK-1(33).
It is important to note that due to the unique characteristics of
alternative macrophage cell lines, they cannot fully replace primary
macrophages in scientific research. Thus, it is essential for
researchers to carefully select suitable cell models when utilizing
alternative macrophage cell lines in their scientific investigations.
4. Fibroblasts
Fibroblasts were first observed by Virchow and Duvall in the mid-19th
century. They are cells present in the fibrous or loose connective
tissues of most mammalian organs(34). Abnormal activation of
fibroblasts, mainly originating from the transformation of normal
resident fibroblasts, as well as epithelial or endothelial cells in lung
tissue, is responsible for many fibrotic diseases, including PF(35).
Myofibroblast foci form due to migration, proliferation, and activation
of mesenchymal cells by activated AECs. Excessive secretion of
extracellular matrix (ECM) proteins by myofibroblasts disrupts pulmonary
homeostasis and structure, causing pulmonary interstitial matrix
sclerosis and pathological matrix deposition(36, 37). In studies
investigating the pathogenesis of PF, fibroblasts are classified into
primary fibroblasts and alternative cell lines. Primary fibroblasts
include human primary fibroblasts and mouse primary fibroblasts.
Alternative cell lines consist of: (1) human embryonic lung fibroblast
cell line (MRC-5 cell line), (2) lung fibroblast cell line (HLF1 cell
line), and (3) mouse embryo fibroblast cell line (NIH3T3 cell line).
4.1 Primary Fibroblasts
Fibroblasts are typically undifferentiated but possess a robust
proliferative capacity. In experimental research, primary fibroblasts
are primarily utilized to examine the relationship between fibroblast
activation, ECM deposition, and PF. Under in vitro culture conditions,
primary fibroblasts maintain good division and proliferation
capabilities.
The excessive activation and proliferation of lung fibroblasts
contribute to extensive ECM deposition; however, the specific mechanisms
driving this process remain unclear. Hence, researchers often employ
fibroblasts to construct pathological models for their investigations.
Li JM et al. observed that fibroblasts deficient in argininosuccinate
synthase 1 (ASS1), isolated from patients with IPF, display an invasive
phenotype characterized by increased migration, proliferation, and
matrix deposition capacities(38). Similarly, Nguyen XX et al. found that
overexpression of hIGFBP5 alters the expression of several structural
and functional macromolecules of ECM in primary fibroblasts(39).
Furthermore, primary fibroblasts have a limited number of passages in
culture, and as time progresses, they gradually lose their functionality
and stability.
4.2 Fibroblast Cell Lines
Due to the limitations of primary cells, fibroblast cell lines have
become the preferred choice for cellular-level research. They offer easy
cultivation, diverse types, and substantial yield, making them valuable
in studying mechanisms such as EMT, activation of signaling pathways,
and fibroblast activation in PF.
MRC-5 cell line was derived from human lung tissue obtained from a
14-week-old male fetus. It exhibits a fibroblast-like morphology and
maintains a normal diploid karyotype during long-term in vitro
expansion. The MRC-5 cell line displays mesenchymal characteristics,
including elongated and spreading morphology, highly dynamic cellular
protrusions, and enhanced migration and invasion potential(40). However,
due to its properties, the MRC-5 cell line is not suitable for studying
a single mechanism alone. For instance, Kim HS et al. demonstrated that
inhibition of the Smad2/3 and ERK pathways reduced fibroblast EMT
activation using a TGF-β1-induced MRC-5 cell model(41). Nonetheless,
being a diploid cell line, MRC-5 cells have limited passaging capacity,
with extended passages leading to cell senescence, which presents
challenges for large-scale production.
HLF cell line comprises normal fetal lung fibroblasts with a normal
karyotype but has a limited lifespan. Recent evidence suggests that
bioactive lipid mediators participate in the pathological processes of
IPF and experimental pulmonary fibrosis. Huang LS et al. treated the HLF
cell line with PF543 and found that the lung fibroblast SPHK1/S1P
signaling axis regulates the expression of mtROS, FN, and α-SMA induced
by BLM or TGF-β through the YAP1 pathway(42). Notably, urocanic acid, an
anti-fibrotic factor in mouse lungs, impairs HLF cell line activity.
Ogger PP et al. reversed IPF metabolic reprogramming by adding exogenous
urocanic acid to cultured HLF cells, reducing proliferation and wound
healing ability(43).
The mouse embryo fibroblast cell line (NIH3T3 cell line) was derived
from NIH Swiss Mouse Embryonic Fibroblasts and is a highly
contact-suppressed continuously passaged cell line. To establish a
sub-strain suitable for transformation analysis, the NIH3T3 cell line
underwent more than 5 rounds of sub-cloning. The NIH3T3 cell line has
been utilized in mechanistic studies of PF. For example, Liu H et al.
established an in vitro model by stimulating NIH3T3 cells with TGF-β1 to
demonstrate that inhibition of the MEK/ERK signaling pathway alleviates
the process of PF(44). In another study, Sun W et al. investigated the
relationship between miR-320a-3p/FOXM1 axis activation and lung fibrosis
using a silica-induced NIH3T3 cell model(45).
It is important to note that fibroblast cell lines undergo continuous
mutations during continuous cultivation, and prolonged multiple passages
can lead to changes in the genotype and phenotype of the cell lines,
potentially affecting experimental results.
5. Co-culture System
In contrast to single-cell culture, cell co-culture involves cultivating
two or more types of cells (from the same or separate tissues) in the
same culture system. Co-culture systems offer several advantages over
single-cell culture as they better simulate the in vivo environment and
allow for comprehensive observation of cell-cell and cell-environment
interactions. In recent scientific studies, two-dimensional (2D) and
three-dimensional (3D) co-culture systems have been utilized to create
in vitro pathology models of PF(46).
5.1 Two-dimensional Co-culture System
In scientific research on PF, researchers mostly use 2D co-culture
systems to investigate underlying mechanisms. Two main constructs are
used: the direct contact co-culture model and the indirect contact
co-culture model.
In the direct contact co-culture system, two or more cells are mixed in
specific ratios and plated on the same interface under predetermined
conditions. This system allows for the demonstration of cellular
interactions. For example, Brookes et al. established a representative
AEC culture model using an air-liquid interface culture with cell lines
exhibiting type I (hAELVis) and type II (NCI-H441) AEC characteristics.
This co-culture system, representing type I and type II lung cells, can
be maintained for over 21 days, providing a promising alternative model
for studying toxic compounds and treatment effects(47).
Indirect contact co-culture involves cultivating different cell types in
such a way that they interact through chemokines within the culture
medium. In this case, chemical signals generated in the culture
environment play a significant role in regulating or influencing cell
behavior, rather than direct physical contact between cells. A
co-culture technique that prevents direct contact between cells is
necessary for studying signaling pathways involved in macrophage M2
polarization. For instance, C. Gan et al. treated fibroblasts with a
conditioned medium from M2 macrophages to determine the effects of
substances released by M2 macrophages on fibroblasts(48).
Compared to cells under in vivo physiological settings, cells grown in
2D culture exhibit distinct growth patterns, shapes, and functions. In
2D culture, cells display flattened growth conditions, undergo aberrant
division, and may lose their differentiated phenotypes, thereby
affecting various cellular functions.
5.2 Three-dimensional Co-culture System
The 3D co-culture system provides a means to study complex cell-cell
interactions within an environment that simulates living conditions.
Although attempts have been made to replicate the in vivo
microenvironment under 2D conditions, cells exist in a three-dimensional
(3D) environment with specific spatial structures. The 3D co-culture
model enables the observation of unique gene expression patterns
resulting from the complex interactions between cells and the
microenvironment in PF, which cannot be fully captured by other
reductionist systems(49).
By utilizing cell culture systems based on biomaterials, researchers can
study changes in cell crosstalk dynamics and mechanical properties of
the microenvironment during the initiation of fibrosis in the distal
lung. Caracena T et al. described a 3D model based on a polyethylene
glycol (PEG) hydrogel in which alveolar type II (ATII) cells started
differentiating into alveolar type I (ATI) cells when embedded with
fibroblasts in a stiff gel, resulting in the highest fibroblast
activation rate among the co-culture conditions(50).
Various cancer organoids have been developed for applications such as
drug screening, radiotherapy screening, genome editing, transplantation,
and oncogene identification(51). Similarly, the use of organoid models
represents a novel and critical step in PF research. Tan Q et al.
generated airway organoids by combining human primary bronchial
epithelial cells, lung fibroblasts, and lung microvascular endothelial
cells under supportive 3D culture conditions(52). Suezawa T et al.
established a new in vitro PF model using alveolar organoids composed of
AECs derived from human pluripotent stem cells and primary human lung
fibroblasts(53).
Undoubtedly, the new three-dimensional cell culture models offer an
attractive approach to overcome the limitations of traditional monolayer
culture. In particular, the 3D co-culture system holds great potential
for simulating physiological and pathological conditions in the human
body. Moreover, the organoid model, as the latest technology for
experimental research on human tissues, is still in the research stage
compared to the traditional model. Its stability, reproducibility,
scalability, and ability to precisely control microenvironmental
conditions are all issues that need to be addressed during its
development(54).