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Clin Transl Sci. 2024;17:e13695.
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https://doi.org/10.1111/cts.13695
www.cts-journal.com
INTRODUCTION
Drug development plays an important role in medical
advancements and directly affects the quality and pro-
cesses of disease prevention and treatment. The complete
process of drug development commences with early drug
discovery, progresses through preclinical studies, human
trials, regulatory reviews, and commercial approval, and
culminating in the availability of the drug on pharmacy
shelves, where postmarketing measures are implemented
Received: 11 July 2023
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Revised: 25 October 2023
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Accepted: 18 November 2023
DOI: 10.1111/cts.13695
REVIEW
Complex invitro model: A transformative model in drug
development and precision medicine
LumingWang1,2
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DanpingHu3
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JinmingXu1,2
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JianHu1,2
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YifeiWang3
1Department of Thoracic Surgery, The
First Affiliated Hospital, Zhejiang
University School of Medicine,
Hangzhou, China
2Key Laboratory of Clinical Evaluation
Technology for Medical Device of
Zhejiang Province, Hangzhou, China
3Hangzhou Chexmed Technology Co.,
Ltd., Hangzhou, China
Correspondence
Jian Hu, Department of Thoracic
Surgery, The First Affiliated Hospital,
Zhejiang University School of
Medicine, Hangzhou 310000, China.
Email: dr_hujian@zju.edu.cn
Yifei Wang, Hangzhou Chexmed
Technology Co., LTD, Hangzhou
310000, China.
Email: wangyifei@accursamed.com
Funding information
National Key Research and
Development Program of China, Grant/
Award Number: 2022YFC2407303;
Major Science and Technology Projects
of Zhejiang Province, Grant/Award
Number: 2020C03058; Research
Center for Lung Tumor Diagnosis and
Treatment of Zhejiang Province, Grant/
Award Number: JBZX- 202007
Abstract
In vitro and invivo models play integral roles in preclinical drug research, evalu-
ation, and precision medicine. Invitro models primarily involve research plat-
forms based on cultured cells, typically in the form of two- dimensional (2D) cell
models. However, notable disparities exist between 2D cultured cells and invivo
cells across various aspects, rendering the former inadequate for replicating
the physiologically relevant functions of human or animal organs and tissues.
Consequently, these models failed to accurately reflect real- life scenarios post-
drug administration. Complex invitro models (CIVMs) refer to in vitro models
that integrate a multicellular environment and a three- dimensional structure
using bio- polymer or tissue- derived matrices. These models seek to reconstruct
the organ- or tissue- specific characteristics of the extracellular microenviron-
ment. The utilization of CIVMs allows for enhanced physiological correlation
of cultured cells, thereby better mimicking invivo conditions without ethical
concerns associated with animal experimentation. Consequently, CIVMs have
gained prominence in disease research and drug development. This review
aimed to comprehensively examine and analyze the various types, manufactur-
ing techniques, and applications of CIVM in the domains of drug discovery, drug
development, and precision medicine. The objective of this study was to provide a
comprehensive understanding of the progress made in CIVMs and their potential
future use in these fields.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© 2023 HANGZHOU CHEXMED TECHNOLOGY CO., LTD and Department of Thoracic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine.
Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.
Luming Wang, Danping Hu, and Jinming Xu contributed equally to this work.
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to monitor potential adverse drug reactions.1 This rigor-
ous and costly investment, averaging 10–15 years and
billions of dollars, underpins the extensive process that a
new drug undergoes prior to official approval for clinical
use.2 Drug development can be divided into preclinical
studies and clinical trials, according to the experimental
stage, objects, and models. Preclinical studies use various
models to identify, screen, and test potential drugs, with
only those drugs exhibiting efficacy and safety advancing
to subsequent clinical trials.3
In vitro cell cultures are useful research tools for mod-
eling human diseases and offer a reproducible and rapid
method for evaluating drug effects and safety. Whereas
conventional 2D- cultured monolayer cells in vitro have
been widely used in recent decades,4 superior 3D- cultured
cell models have been developed to achieve greater func-
tion and state of cultured cells. However, both these sim-
ple in vitro models do not fully replicate the complex
tissue environment or the physiological and pathological
processes that occur in the human body, and thus cannot
mimic the physiologically relevant functions of human or
animal organs and tissues.
Complex in vitro models (CIVMs) have been defined
as systems in a 3D multi- cellular environment within
a biopolymer or tissue- derived matrix, which incorpo-
rates primary or stem cell- derived cells, immune system
components, and mechanical factors, such as stretch or
perfusion, or at least two of these elements.5 CIVM en-
compasses frontier 3D cell culture, including organoid
technology, organ bud culture, spheroid culture, tissue
slice culture, 3D bioprinting, and hydrogel- based tissue
engineering. Another representative technique of CIVMs
is microfluidic technology, which enables the precise ma-
nipulation of fluid flow to replicate blood circulation and
further simulate drug absorption, distribution, metabo-
lism, and elimination by fabricating interconnecting mi-
crochambers and microchannels. Consequently, CIVMs
such as patient- derived organoids (PDOs),6 and organ- on-
chip technology7 are now more frequently used in disease
research and drug screening. Compared to normal invitro
2D cultured cell models, these CIVMs emulate the mi-
croarchitecture and functional characteristics of native or-
gans and fully reflect the complexity of the drug response,
thereby offering more accurate results for drug efficacy.
Meanwhile, the “FDA Modernization Act 2.0” passed by
the U.S Senate in 2022 authorized the use of certain al-
ternatives to animal testing, including cell- based assays
and computer models, to investigate drug safety and effec-
tiveness, eliminating the need for animal studies as part
of the process to obtain a license for a biological product
that is biosimilar or interchangeable with another biolog-
ical product (https:// www. congr ess. gov/ bill/ 117th - congr
ess/ senat e- bill/ 5002? q=% 7B% 22sea rch% 22% 3A% 22FDA+
Moder nizat ion+ A c t +2. 0% 22% 7D& s= 10& r= 1 ).8 This sig-
nals a major shift in that animal tests are no longer indis-
pensable, and CIVM may be an alternative to drug safety
regulation.
CIVM has emerged as a promising approach for mod-
eling disease, development, and homeostasis of various
human organs and it plays an increasingly important
role in drug screening and medicinal development.
Understanding CIVM may help in the development of
new drugs and precision medicine. In this review study,
CIVM was introduced based on the progress of organ-
oid technology, tissue slice technology, and microfluidic
organ- on- chip technology (Figure1). The applications of
CIVM based on these technologies are also summarized to
provide a reference for the development of CIVM.
3D CELL CULTURE SYSTEM
REPRESENTED BY ORGANOIDS
An organoid is a “3D structure derived from either pluri-
potent stem cells (PSCs), neonatal tissue stem cells or
adult stem cells (ASCs)/adult progenitors, in which cells
spontaneously self- organize into properly differentiated
functional cell types and progenitors, and which resem-
ble their invivo counterpart and imitating some functions
of the organ.”9 Although various 3D culture technolo-
gies have been rapidly established and widely used after
the successful isolation of Matrigel in the 1980s,10 the
first single stem cell- derived organoid, which has special
characteristics of self- proliferation capability and differ-
entiation potential to form organ- like structures, was not
established until Sato etal. reported that Lgr5+ intestine
stem cells self- organized into intestinal crypt- villus struc-
tures without a mesenchymal niche in Matrigel in 2009.11
This technique marked significant revolutionary progress
in enabling strict recapitulation of invivo cell signatures
and has since emerged as a powerful tool for maintaining
epithelial cells in a near- native state.11
According to the literature, organoids may be gener-
ated through induced differentiation of stem cells. With
advancements in human stem cell culture, organoids from
various cellular sources, and species have been generated
successfully.12 The three fundamental elements of organ-
oid formation are media composition, cells, and the ma-
trix (Figure2).
Media composition for cultivation of
organoids
For the development and growth of organoids, media
compositions that recapitulate the invivo stem cell niche
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CIVMS AND THEIR APPLICATION IN BIOMEDICAL
signaling pathways that sustain stem cell function, drive
their expansion, and eventually their differentiation, are
essential.13 Most organoids are derived from stem cells,
which require exposure to specific morphogens at defined
timepoints to activate the desired developmental signal-
ing pathways and trigger self- organization. This deter-
mines the importance and diversity of organoid media
composition.
Various types of organoids require specific optimal
media compositions, and only when components are pro-
vided exogenously can organoids proceed with appropriate
generation and maintenance. Mouse optic cup organoids
predominantly rely on endogenous signals, eliminating
the need for specific exogenous signals and are cultured
in a serum- free medium with minimal growth factors.
The formation of its uniform neuroepithelium, followed
by self- patterning mechanisms that specify spatially sep-
arated domains of the neural retina, retinal pigmented
epithelium, and morphogenesis, all proceed appropriately
and follow its default developmental trajectory.14 However,
the initial cellular system of most organoids lacks essen-
tial components to undergo the desired self- organization
process. The media composition of the majority of organ-
oids requires supplementation with specific exogenous
signals to ensure their intended and correct developmen-
tal trajectory. Takasato etal.15 reported a protocol for the
generation of kidney organoids from human embryonic
stem cells (hESCs). Supplementation with specific growth
factors, such as BMP4, activin A, FGF9, BMP7, and RA,
was required to stimulate hESC differentiation to recipro-
cally induce kidney progenitor populations, which would
then self- organize into kidney organoids without further
factors. Some organoids, such as gastric organoids16 and
human fetal- like forebrain organoids17 require specific
exogenous stimulation throughout the derivation process.
Supplementation of media composition may vary depend-
ing on the specific type of organoid. The contents of addi-
tional specific exogenous signals, such as growth factors,
signaling agonists, and inhibitors also vary in organoid
cultures. For example, Wnt/β- catenin signaling, TGF- β
signaling, and EGFR2 signaling- associated components,
such as Wnt- 3A, BMP- 4, Activin A, EGF, FGF- 10, FGF-
7, HGF, and SB 431542, are included in the list of media
supplements.18,19
FIGURE Schematic diagram of common invitro models and typical CIVM (by Figdraw UWTTI9f63b). Cells used to establish invitro
models are derived mainly from cell lines or primary cells isolated from tissues. Single- cell suspension can fabricate various invitro models
such as organoids, spheres, and microfluidic organ chips with the use of different culture technologies. Tissue slices can be formed from
total tissue by tissue slicing. CIVM, complex invitro model.
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Cell resources for organoid generation
Two types of stem cells are used in organoid culture.
One is embryonic pluripotent stem cells (ESCs) or ESC
states, such as induced pluripotent stem cells, which are
responsible for embryonic organ development. These
cells have been successfully used to differentiate into
and form a broad range of tissue- specific organoids.20,21
The other type comprises ASCs or organ- specific resi-
dent stem cells, which are important for maintaining
mature organ homeostasis and facilitating regenera-
tion.22 These stem cells have already demonstrated their
ability to grow into organoids invitro after obtaining the
proper extracellular matrix (ECM) and molecular clues.
For example, mouse intestinal organoids are initially
generated from Lgr5 + ASCs in the absence of a mesen-
chymal niche.11 In summary, most organoids from the
surface ectoderm lineage or endodermal lineage, rep-
resented by glandular tissues, are derived from ASCs,
dissociated adult tissues, or PSCs. Neuroectodermal,
cerebral, and mesodermal kidney organoids are exclu-
sively derived from PSCs.23
Generally, organoids can be obtained through the der-
ivation of a single cell type or co- culture of separate pre-
established cell types. However, the starting conditions
and research potentials may differ. Organoids from single-
cell types, such as the reported optic cup or small intestine
organoids, typically undergo an initial cell expansion step
before self- organization,24 whereas the co- culture meth-
ods of organoids initiate from pre- differentiation and an
appropriate proportion of each cell type.25 Mesenchymal
stem cells (MSCs) have previously been shown to contrib-
ute to the contraction force that drives self- organization
and are indispensable for the co- culture approach of
organoid formation.26 The scope of applicability of an
organoid as a biological model system varies due to the
differences in the formation process. Organoids derived
from a single cell type that undergoes cell differentiation
and the simultaneous generation of different cell types
may offer additional insights for organogenesis. Organoids
from co- cultured pre- differentiated cells establish distinct
cell identities. Their self- organization primarily involves
cell sorting and subsequent architectural rearrangements,
enabling them to comprehensively capture the transient
FIGURE Three key points of organoid formation (by Figdraw, ID: OIYAI1111a).
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CIVMS AND THEIR APPLICATION IN BIOMEDICAL
developmental interactions between different progenitors
during organoid formation.26
Matrix for organoid generation
The last element is the matrix, which supports cell growth
and adherence. Matrigel, a natural solid ECM purified
from Engelbreth- Holm- Swarm mouse sarcoma,27 is widely
used to promote intestinal, cerebral, gastric, and mammary
gland organoids.20,28 Matrigel and other similar animal-
derived hydrogels, such as collagen type I matrices, mimic
basement membranes and support cell adherence and
growth. These natural matrices contain a complex mixture
of ECM components and growth factors, facilitating effi-
cient cell growth and differentiation. However, these ma-
trices exhibit batch- to- batch variability, making it almost
impossible to analyze and control their exact compositions
and contents. This increases the difficulty in controlling the
culture environment and reproducing experimental results.
Chemically defined hydrogels have a definite composition
and therefore represent promising candidates. Synthetic
hydrogels, such as alginate hydrogels,29 hyaluronan- gelatin
hydrogel,30 methacrylate gelatin,31 polyethylene glycol,
and polyacrylamide32 may support the culture of intestinal,
pancreatic, neural, colorectal cancer, prostate cancer, glio-
blastoma, and hepatic organoids and retain the expression
profile of key markers.33,34 Further, these synthetic hydro-
gels allow the biochemistry and mechanics of the cultural
environment to be controlled. However, they lack bioactiv-
ity and need to be customized to meet the specific require-
ments of different organoids.
With considerable advances in organoid culture tech-
nology, Matrigel is no longer an essential element. Co-
culture- based organ buds are considered non- matrix
(Matrigel) organoids because they are also tiny self-
organized structures. These organ buds are assembled
from various PSCs through intercellular and ECM interac-
tions. The first reported co- culture- based organ buds were
liver buds, which were created by mixing tissue- specific
progenitor cells derived from PSCs, endothelial cells
(ECs), and MSCs.25 Through extensive research, organ
buds for different organs, such as the kidneys, pancreas,
intestines, heart, lungs, and brain, have been developed.35
Another strategy for growing organoids involves sus-
pension cultures. This method has already been adopted for
deriving optic cups, cerebral, cerebellar, kidneys, acinar/
ductal, and hippocampal organoids.36,37 The suspension
method involves growing the organoids inside thermo-
formed microwell arrays and promoting their controlled
growth under matrix- reduced conditions, with Matrigel
being used only as a medium supplement and not a scaf-
fold, or even under no- matrix conditions. Kumar et al.
generated human PSCs- derived kidney micro- organoids
using a modified suspension culture method. Briefly, cell
suspensions in low- adhesion culture plates were swirled at
low- speed (60 rpm) to form cell aggregates in the presence
of differentiation media containing 0.1% polyvinyl alcohol
and methylcellulose. However, the formation of variable
patterns of dysplasia present in the prolonged suspension
culture resulted in the loss of a functional proximal tubule,
reduced expression of many kidney marker genes, and fi-
brotic lesions followed by apoptosis of the epithelium and
deposition of the ECM (α- SMA).37
Human colorectal epithelial organoids38 have also been
generated using low- viscosity matrix suspension culture.
Organoids and toroids grown in a culture medium contain-
ing 5% Matrigel and 10 μM Y- 27632 appear to reduce the cost
of organoids and fitting high- throughput drug screening
due to their ability for rapid expansion. However, the ECM
is a fundamental, core component of all tissues and organs.
The stiffness and biochemical substances of the matrix are
important components of the microenvironment, and the
matrix is involved in various biological behaviors.39 Cancer
cells, the heterogeneous collection of infiltrating and res-
ident host cells, secreted factors, and ECM together make
up the tumor and tumor microenvironment.40 Increased
matrix stiffness reportedly has profound effects on tumor
growth and metastasis. The absence of a matrix when es-
tablishing organoids invitro may compromise mimicry of
the tumor microenvironment invivo.41 Thus, careful selec-
tion of matrix- reduction or non- matrix methods for organ-
oids is vital to maintain the accuracy of drug screening.
TISSUE SLICING TECHNOLOGY
AND TISSUE SLICES
The tissue slice culture prototype was first proposed by Dr.
Harford's team and involved cutting the tissue into small
pieces.42 These tissue slices can be separated directly from
normal organs and tumor tissues from humans and ani-
mals, providing the natural advantage of maintaining com-
plex structural features and microenvironments in vitro.43
However, tissue slices cannot be maintained under long-
term culture or expansion in vitro, with the culture period
typically lasting 1 to 2 weeks only.44 These characteristics
make tissue slices suitable for the short- term study of disease
mechanisms and prediction of single- use curative effects
invitro, especially for immune- related drugs or therapies.
Fabrication methods of tissue slices
Tissue slices can be fabricated using two methods. The
first method is manual cutting, which typically produces
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millimeter- thick samples with irregular shapes. It is diffi-
cult to ensure the survival of cells at the center of the sam-
ple and the process lacks repeatability.45 The other method
is vibratome technology, which can generate slices from
tens to hundreds of microns. Tissue slices generated by
the vibratome are precisely cut to obtain a relatively regu-
lar shape, making them superior for culture and testing.46
Therefore, the second method is most commonly used to
generate tissue slices.
Equipment, such as a Krumdieck microtome, vibratome,
and compresstome can used for tissue slicing. These equip-
ments rely on the vibration of the blade during slicing to
reduce pressure and stress on the fresh tissue sample.47 The
Krumdieck microtome, vibratome, and compresstome dif-
fer in the thickness of the tissue that can be cut and the
way the sample is sliced. The Krumdieck microtome gen-
erates slices ranging from ~100 to 500 μm, with a slicing
rate of 3–4 s per slice. Before slicing, the tissue is cut into
a cylindrical shape and placed into a sample hole, which
is then fixed perpendicularly to the microtome blade. To
regulate slice thickness, the distance between the blade
and the screw- controlled limit plate is adjusted during the
slicing process.48 The compresstome shares a similar slice
generation process to the Krumdieck microtome but the
slice thickness ranges from ~30 to 1000 μm. The VT1200 S
vibratome (the most commonly used vibratome) produces
slices of a similar thickness to that of the compresstome.
The tissue is immobilized on the sample table of the Leica
VT1200 S vibratome before slicing, and the slice thickness
is controlled by lifting the sample table.
Culture of tissue slices
Culture slices is followed by generation. The vitality and
specific functions are the basis for further biological ex-
periments. Slice thickness, culture medium, and specific
culture methods are important considerations.
Research has determined that tissue slice thickness of
100–300 μm is optimal due to the limitation of oxygen and
nutrient penetration, which typically spans 10–20 cell lay-
ers (~150 μm). Moreover, slices thinner than 100 μm may re-
sult in a high percentage of injured cells caused by slicing.45
Slices of 200–300 μm thickness are the most commonly re-
ported in publications to date. In addition, the media used
for these tissue slices are easier to use than that for organoid
culture, considering that they do not require differentiation
or self- organization processes. The culture medium is simi-
lar to that of homologous cell culture. However, the culture
of immune cells requires the appropriate addition of IL- 2,
especially when testing immunomodulatory drugs.49 For
specific culture methods, tissue slices are usually placed
on a supporting tool, such as a Millipore filter insert, to
navigate the air- liquid interface culture.50 However, these
methods still result in intra- slice gradients. Microfluidic
techniques have been used to overcome this limitation.
The perfusion air culture system provides a continuous and
controlled oxygen medium and drug supply.51
MICROFLUIDIC TECHNOLOGY
AND MICROFLUIDIC CHIPS
FOR CIVM
The microfluidic technique involves the precise manipula-
tion of fluids using microscale device technology first de-
veloped by the semiconductor industry and later expanded
upon using microelectromechanical systems field.52 These
techniques are based on the combined principles of phys-
ics, chemistry, biology, fluid dynamics, microelectronics,
and material science, and are characterized by engineered-
manipulated fluids at submillimeter- scales.53 Precisely-
controlled fluids and particles enable the manipulation
and analysis of cultured cells. Researchers recognize the
significance and advantages of medicine and engineer-
ing across disciplines and concur that the development of
comprehensive microfluidics could potentially solve issues
in biology and clinical research. However, the integration
of novel microfluidic techniques in mainstream biologi-
cal research has not matched the initial enthusiasm in the
field. Reports indicate that microfluidic technology has
been primarily used for diagnostic applications and the
manipulation of blood samples in mainstream biomedical
research over the past decade, despite its potential in areas
such as nanoparticle preparation, drug encapsulation, de-
livery, targeting, cell analysis, diagnosis, and cell culture.53
Fortunately, the development of cell culture technology has
increased the demand for drug development and precision
medicine. The adoption of novel microfluidic techniques
in constructing invitro models has progressed rapidly and
has become the primary application of microfluidic tech-
nologies because the microfluidic system integrates the
functions of driving, manipulating, monitoring, reacting,
detecting, and analyzing. This allows for the simultaneous
optimization of culture conditions, treatment, and detec-
tion.54 Microfluidic organ chips can be categorized into
single- organ chips and human- on- chips based on their tar-
get function, degree of complexity, and the types of organs
in the system (Figure3).
Microfluidic organ- on- chips (single- organ
chips)
The use of microfluidic devices has considerably ad-
vanced the field of CIVMs because of their ability to
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CIVMS AND THEIR APPLICATION IN BIOMEDICAL
FIGURE Typical microfluidic organ chips (by Photoshop). (a) Diagram of a sandwiched human liver- chip fabricated by primary
human hepatocytes within an extracellular matrix on a porous membrane.69 The whole channel was divided into an upper parenchymal
channel and a lower vascular channel by the sandwiched structure. (b) Diagram of a physiologically inspired two- organ chip.110 Liver
spheroids and bronchial tissue were seeded in the right and middle chambers separately to mimic the MucilAir culture and liver model.
The medium in the left chamber progressed through the MucilAir culture chamber and then through the liver chamber. (c) Diagram of
a physiologically inspired four- organs- chip.70 This chip consists of a surrogate blood circuit (red) and an excretory circuit (orange). The
surrogate blood circuit includes three chambers simulating the liver, brain, and the intestines, whereas the excretory circuit includes two
chambers simulating the glomerulus and renal tubule.
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mimic the fluid environment of native living cells and
allow for the co- culture of complex cellular compo-
nents.55 Microfluidic organ- on- a- chip or human- on- a-
chip is the most typical application and can potentially
replace animal testing.56 The concept of the organ- on-
a- chip system originated from attempts to control and
optimize cell culture invitro by applying various micro-
fluidic systems. The aim is to mimic the key organotypic
cellular architecture, functionality, and environment on
a smaller scale for the purposes of disease modeling and
drug screening.57
The field of microfluidic organ- on- chip has rapidly pro-
gressed since the lung- on- a- chip model was built on Huh's
early work by the Takayama Group in 2010.1,7 This lung- on-
a- chip model had two microfluidic channels separated by
a porous membrane on which lung alveolar and capillary
cells could be co- cultured. This model provided research-
ers with the opportunity to decipher the breathing mecha-
nisms occurring in the alveoli, the capillary interface of the
human lungs, environmental effects on lung cells, and the
pathological mechanisms of various pulmonary or other
respiratory diseases invitro.58 To date, organ- on- chips for
investigating disease progression and analyzing adverse
drug reactions include liver chips, lung chips, heart chips,
kidney chips, pancreas chips, gut chips, bone and bone
marrow chips, brain chips, reproductive organ chips, and
muscle chips have been successfully developed.
Biological barriers are important for the maintenance
of organ homeostasis and are essential for drug testing
and disease modeling.59 In vitro models of functional
biological barriers, such as blood–brain barrier (BBB),60
have previously been created through the development
of 3D cell culture techniques and microfluidic organ- on-
chip technology. Yu et al.61 developed 3D microfluidic
BBB chips by co- culturing rat primary brain microvas-
cular ECs, pericytes, and astrocytes from neonates in
a collagen matrix. Similarly, Kim etal.62 constructed a
simplified 3D co- culture- based BBB model within 30 min
using immortalized human brain ECs and immortalized
human astrocytes mixed with Matrigel. This simplified
3D co- culture- based BBB model, comprised solely of im-
mortalized brain ECs, blocked the penetration of dextran
molecules with various molecular weights, remained du-
rable and impermeable even under BBB- degrading con-
ditions, and rapidly formed tight junctions.
Microfluidic human- on- chips
Currently, the more advanced and complex “body- on-
chip” or “human- on- a- chip,” mirrors the physiology of the
entire human body for drug pharmacokinetic and pharma-
codynamic analyses. This technology uses interconnected
invitro microfluidic devices to model human tissues and
was established using multiple organs- on- chips.63
Building on the concept of single- organ chips, more
complex and advanced multi- organ chips integrating
multiple organ units have been constructed. Multi-
organ chips can mimic individual organ functions and
have further advantages in integrating the functions of
the constituted single- organ part, such as drug absor-
bance in the gut compartment, drug metabolism in the
liver compartment, and drug elimination in the kidney
compartment. It mimics multiple systems that interact
invivo thereby enabling comprehensive studies. For ex-
ample, Pires de Mello etal.64 developed a three- organ
heart–liver–skin system. A skin surrogate (Strat- M
membrane) was used to mimic the absorption processes
of the topically administered drugs to be tested and as-
sess their toxicity. The results indicated that the heart–
liver–skin three- organ system can be used to assess
potential drug toxicity from dermal absorption, as well
as evaluate transport dynamics through the skin. Shi65
developed a BBB- glioma microfluidic chip (BBB- U251
chip) composed of a BBB unit and glioma cells. The BBB
unit was formed by the co- culture of primary human
brain microvascular ECs, pericytes, and astrocytes.
The BBB- U251 chip displayed selective permeability to
FITC- dextran with various molecular weights and three
model drugs with different permeabilities. This glioma
model could replicate the barrier function of the human
BBB as well as the glioma microenvironment.
Human- on- chip confers the advantage of investi-
gating inter- organ communication in response to drug
challenges at the human level, which provides drug de-
velopment activities with the potential to lower the cost of
preclinical studies and increase the rate of drug approval
by introducing human phenotypic models early in the
drug discovery process.64 It also provides a novel approach
to rare disease research and orphan drug development.66
For instance, researchers at Cornell University created a
13- organ recirculating system. This microfluidic cell cul-
ture device had pumpless 14 chambers (13 organs) that al-
lowed for separation between the barrier and non- barrier
cell culture types, and supported all cell types maintained
for up to 7 days.67
Key parameters for organ- chips
One of the most important purposes of microfluidic organ
chips is to provide cultured cells and tissues with a media
flow similar to the circulatory system and fluid extracel-
lular environment invivo. Culture chambers and liquid
control systems are critical constituents for achieving
this. The design and regulation between the volume and
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CIVMS AND THEIR APPLICATION IN BIOMEDICAL
runner of the chamber and the input and output of the
medium lead to variations in the medium flow rate which
may influence the culture of cells or tissues.68 Here, we
summarize some culture parameters.
The design of chips varies greatly to fit different ap-
plications but have similar design logic; culture cham-
bers and fluid chambers are essential structures and the
key point of distinction. The two most common designs
are strip- type and round- hole- type culture chambers.
For instance, the classical chip of Emulate Inc. has two
strip- type chambers (top and bottom channels) separated
by a porous poly dimethylsiloxane (PDMS) membrane
(Figure3a). The cells are seeded on both sides of the
PDMS membrane.69 The classical chip of TissUse GmbH
has a round hole linked by different microfluidic chan-
nels (Figure3b). Cells, organoids, and other samples are
cultured in a round hole.70 As chips have similar culture
chambers, they can achieve different functions and can
be differentiated based upon the fluid flow direction and
its cause. A classical type of flow is generated by pressure
from a pump, enabling unidirectional fluid flow from the
input to the output side of the culture chamber. Another
type of flow is generated by sloshing the liquid. A chip
plate containing an appropriate amount of medium is
placed on a shaking device that moves similar to a see-
saw. The fluid flows from a higher position to a lower
position, generating a unidirectional flow, and then re-
versing as the shaking device changes position.
Various cells and tissues experience different fluid flow
stresses invivo; therefore, they have different optimal shear
stresses and tolerance ranges.71 Consequently, the flow rate
is an important consideration because it can influence the
fluid flow stress experienced by the cultured samples.
Materials used in microfluidic organ chips
The selection of materials for microfluidic chips plays
a vital role in facilitating the utilization of microfluidic
models in drug development, as it directly impacts the
compatibility and interaction of drugs within these mod-
els. The decision regarding materials in microfluidics is of
utmost importance, with biocompatibility being the pri-
mary consideration due to its significance in supporting
the cultivation of biological cultures within microfluidic
organ chips. Subsequently, the interaction with drugs or
detection reagents must be taken into account as a sec-
ondary consideration. Materials with high binding affinity
or drug adsorption properties with drugs and detection re-
agents can interfering with the results of drug screening or
basic medicine research. Finally, the optical transparency,
fabrication difficulty, mechanical properties, and cost also
need to be considered.72
The PDMS is extensively utilized in microfluidic de-
vices due to its transparency, ease of fabrication, and
biocompatibility. Its diminished affinity for hydrophobic
drugs renders it suitable for drug screening and delivery
investigations. Nonetheless, PDMS has the propensity
to adsorb hydrophobic drugs, leading to their gradual
depletion from the solution.73 Glass, on the other hand,
is a frequently used material that exhibits remarkable
chemical resistance and optical transparency. Its limited
drug adsorption properties render it appropriate for drug
investigations that necessitate precise maintenance of
drug concentrations.74 Polymethylmethacrylate, a cost-
effectiveness transparent material possessing favorable
optical attributes and low drug adsorption properties, is
widespread used in microfluidic devices and drug compat-
ibility studies. Besides, materials like natural biomaterials
(represented by gelatin, alginate, and collagen), PLGA,
etal. are used to fabricate microfluidic chips.72
APPLICATION OF CIVMS IN DRUG
DEVELOPMENT AND PRECISION
MEDICINE
Positioning of CIVMs in drug discovery
and drug development
Drug discovery focuses primarily on identifying and de-
veloping new therapeutic targets and potential drug can-
didates. This involves initial research and exploration to
identify molecules or compounds that may potentially
treat specific diseases, including activities such as target
identification, lead- compound identification, and early-
stage testing in laboratory settings. Drug discovery is pri-
marily conducted by scientists in academic and industrial
research laboratories. Once a promising drug candidate is
identified during the drug discovery phase, it progresses
to the drug development stage, which involves extensive
testing and evaluation to determine its safety, efficacy,
and optimal dosage. This stage includes preclinical stud-
ies, clinical trials (phases I, II, and III), regulatory approval
processes, and postmarketing surveillance. Drug develop-
ment focuses on refining and optimizing candidate drugs;
understanding their pharmacokinetics, toxicity, and po-
tential side effects; and gathering sufficient clinical evi-
dence to support their approval and commercialization.
Animal and invitro cell experiments have long been
integral to life science research. Experiments in the drug
discovery and development stages should be conducted on
animals or in vitro 2D models that exhibit similar habits
or characteristics that are relatively consistent with those
of humans to study ontogeny, disease pathogenesis, and
drug treatment effects. This is an essential step for new
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WANG etal.
drugs before they are permitted to enter clinical trials.
Only drugs with proven safety and efficacy, following a
series of animal studies, are approved for clinical trials.2
Millions of animal models have been established and
are widely used in basic medical research. Small mam-
mals, such as mice, rats, and zebrafish, as well as large
mammals, such as swine and nonhuman primates, are
popular and valuable in preclinical testing due to the sim-
ilarity of their phenotypes, organ sizes, and physiologies
to those of humans. Animal models are crucial for bio-
medical research and drug development. The translation
of animal models into human subjects remains unpre-
dictable owing to species variation and inherent hetero-
geneity, which may lead to unexpected results. Moreover,
ethical concerns and the necessity of animal models are
frequently mentioned by the scientific community.75
The tenet of the three R's (Replacement, Reduction, and
Refinement) and animal welfare in animal research are
emphasized throughout the animal experimental process.
There is an apparent trend toward greater standardization
and reduced reliance on animal models under the guid-
ance of the three R's.
The 2D models have played a significant role in the
past decades, not only in basic medical research, but
also in drug discovery and development. A cancer cell
line panel consisting of dozens of human cancer cell
lines derived from different types of cancers was used for
high- throughput screening of drugs prior to selection for
further preclinical assessment in xenograft models. Using
a large panel of 77 colorectal cancer cell lines providing
sufficient cell lines to represent each genetically defined
subtype in primary cancers, a clear relationship between
5- fluorouracil sensitivity and mismatch repair status in a
subset was shown.76 Whereas 2D models are classic and
contribute significantly to scientific research, they do not
reappear in physiological tissue environments or invivo
physiology and pathology in the human body. Therefore,
they cannot mimic the physiologically relevant functions
of human and animal organs and tissues. In contrast, sim-
ple 3D- cultured cell lines were developed as an optimized
version of 2D culture. An investigation indicated that oxy-
resveratrol (OXY), an anticancer therapeutic agent, inhib-
its breast cancer cell proliferation. Although, OXY is an
effective cytotoxic agent in 3D tumor models, its effect in
2D models is less pronounced.77
Another study tested the viability of 3D or 2D cultured
malignant pleural mesothelioma (MPM) cell lines and
analyzed the antitumor effects of cisplatin (CPDD) and
pemetrexed (PEM). The results demonstrated that PEM
alone and PEM combined with CPDD most effectively
reduced MPM cell viability. The 3D culture models are
important and superior in cancer studies and invivo- like
drug testing.78 Considering that cells live in a complex
microenvironment with various cell types, extracellular
matrices, and physical stimuli, the aforementioned simple
invitro models are not ideal for accurately representing
the drug response in the human body.
Research on humans cannot be conducted directly
until sufficient evidence confirms the safety and efficacy
of a drug or treatment. Therefore, advanced models are re-
quired to obtain evidence of safety and efficacy at a lower
cost and in a shorter time. CIVMs are platforms to depict
body reactions against drugs and may reduce investments
and shorten the time for drug discovery and drug testing
both in drug development and precision medicine owing
to its superior in applying human physiology and neuro-
pathology at an organ or whole- body level, compared to
normal 2D cell and animal models. In August 2022, the
US Food and Drug Administration (FDA) granted ap-
proval for a clinical trial Investigational New Drug (IND)
application for Sanofi's drug Sutimlimab. This approval
marks a significant milestone as it is the first time that the
FDA has approved a clinical trial IND application based
solely on preclinical efficacy data obtained from human
organ- on- a- chip studies, in conjunction with existing
safety data (https:// www. nih. gov/ news- events/ news- relea
ses/ resea rcher s- creat e- 3- d- model - rare- neuro muscu lar-
disor ders- setti ng- stage - clini cal- trial ). Additionally, in the
same year, the United States enacted legislation that elim-
inated the stringent mandate for animal testing prior to
FDA approval for new clinical trials.
Representative CIVMs, such as organoids, organ-
on- chips, or human- on- chips, and tissue slices usually
appear in publications on disease research as the prin-
cipal focus of disease modeling. These technologies and
products differ from each other, but have the potential
for complementarity and collaboration for significant
advancements. For example, organoids usually have
self- assembled structures and multiple- cell components
but lack mechanical factors, such as perfusion.79 Tissue
slices have complex immune system components that
are highly consistent with the microenvironment invivo
but are limited in nutrient transport and long- term cul-
ture, and mimic interstitial flow.80 These deficiencies
limit the ability of a CIVM to reach a stage that is more
consistent with the original tissue. Fortunately, the ap-
plication of microfluidic culture technology provides an
opportunity to overcome these limitations, enhancing
the culture efficiency and interstitial flow stimulation
for organoids and tissue slices.81
These advantages make CIVM not only highly valu-
able in studying disease mechanisms and target screen-
ing during the drug discovery stage, but also superior in
evaluating safety and efficiency during the drug devel-
opment stage. Meanwhile, drugs that can be evaluated
are limitless in nature. In addition to innovative drugs,
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CIVMS AND THEIR APPLICATION IN BIOMEDICAL
CIVMs have further opportunities for the efficiency and
safety evaluation of generic drugs and excipients, as well
as expanding the indication of drugs. The absorption,
metabolism, and toxicity of ginsenoside compound K,
a carbohydrate drug with numerous biological activi-
ties and physiological functions, have been successfully
investigated using single- organ and multi- organ chips
based on intestinal, vascular, liver, and kidney chips.82
Liu et al. analyzed 870 liver chips to determine their
ability to predict drug- induced liver injury caused by 27
known hepatotoxic and non- toxic drugs. With a sensi-
tivity of 87% and a specificity of 100%, the liver chips
outperformed conventional models. This suggests that
widespread acceptance of CIVMs, such as organ chips,
could decrease drug attrition and generate billions an-
nually for the pharmaceutical industry through in-
creased drug development and small- molecule research
and development productivity.69
CIVMs for the study of disease
mechanisms and identification of drug
targets in drug discovery
Organoids, as a component of CIVM, can serve as models
of human diseases. A wide range of organoid- based dis-
ease models that replicate genetic diseases, host–pathogen
interactions, and cancer have been developed and have
certain well- known pathological features. For example,
Welm etal.83 reported human xenograft- derived organoid
(PDXO) cultures from patients with endocrine- resistant,
treatment- refractory, and metastatic breast cancers. These
PDXO models have demonstrated high fidelity to their
original tumors, and their drug responses are concordant
with invivo responses and could predict drug responses
in patient- derived xenografts. Precancerous pathologies
encompassing endometrial hyperplasia and Lynch syn-
drome organoids, patient- derived cholangiocarcinoma
organoids, ovarian cancer organoids, and patient- derived
upper tract urothelial carcinoma organoids that capture
disease diversity have also been established and will serve
as powerful research models to aid in drug screening and
discovery.84
Microfluidic organ chips containing cells or organs
represent an alternative method for drug discovery.
Sengupta et al.85 established a breathing lung- on- chip
system composed of immortalized human alveolar epi-
thelial cells that represented both AT1 and AT2 charac-
teristics, thereby presenting a valuable in vitro tool for
studying inhalation toxicity, testing the safety and effi-
cacy of drug compounds, and characterizing xenobiotics.
Moreover, it has potential applications in coronavirus
disease 2019 research.86
CIVMs for the study of preclinical
evaluation in drug development
Disease models, such as PDOs or tissue slices, can serve as
excellent tools for evaluating drug efficiency. Hong etal.
fabricated tumor tissue slices from patients with clear cell
renal cell carcinoma (ccRCC) to evaluate the antitumor
effectiveness of a V- domain Ig suppressor of T- cell acti-
vation (VISTA) inhibitor, which is a candidate immune
checkpoint inhibitor (ICI).87 The results indicated that all
tested samples responded to the anti- VISTA monoclonal
antibody and produced TNF- α, with 20% of ccRCC sam-
ples showing a synergistic effect when treated with a com-
bination of VISTA and PD- 1 inhibitors.
Microfluidic organ chips are advantageous for study-
ing cell interactions and testing drug candidates for dis-
eases. Organ- on- chips (especially multi- organ chips or
human- on- chip models) are superior for assessing drug
toxicity in healthy human organs and the efficiency
of disease by considering drug absorption and meta-
bolic processes in the body.88 A collagen- based 3D pri-
mary human hepatocyte (PHH) model was constructed
using the biomimetic array chip. This model showed
improved and stabilized liver functionality in terms of
cell viability, albumin, and urea production compared
with the 2D PHH model and showed a higher sensitivity
for predicting the hepatotoxicity of clinical drugs, indi-
cating its potential for risk assessment of drug- induced
hepatotoxicity.89 Heart- on- chip and cardiac organoids
have been used to predict cardiotoxicity of drugs.90 An
optimized microfluidic chip design consisting of inter-
connected compartments was presented. Such a sim-
plified tandem system is a liver- kidney- on- chip model
that includes a liver compartment containing hepatic
cells that grow abundantly in microfluidic conditions
and stably express metabolism- related biomarkers and
a glioblastoma compartment. The biotransformation
and toxicity of Aflatoxin B1 and benzo(a)phapyrene, as
well as the interaction with other chemicals, were suc-
cessfully investigated using this system, demonstrating
that the toxicity and metabolic response to drugs can
be evaluated in advanced interconnected multi- organs
chips.91 Intestine- liver- on- chip systems have also been
developed and used to predict oral drug administration
and first- pass metabolism invitro as an emulation of the
first- pass mechanism occurring invivo.92 In addition, a
four- organ chip integrated with sequentially connected
intestinal, liver, skin, and kidney compartments with
stable homeostasis across different organ compartments
was developed to test the heart and liver toxicity of acute
and chronic drug exposure.93
Biological barrier chips are special well- developed
organ chips. Researchers modeled neuroinflammatory
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WANG etal.
conditions that compromised BBB functionality and ob-
served the protection of the BBB after treatment with
the glucocorticoid drug dexamethasone.61 In addition,
in vitro BBB- glioma microfluidic chip model could re-
produce the high level of barrier function of the invivo
human BBB and allow for the establishment of the gli-
oma microenvironment.65 The drug efficacy of six po-
tential anti- glioma components from traditional Chinese
medicine was evaluated by delivery into the blood chan-
nel of the chip. The data closely resembled in vivo data
from the traditional Transwell model, indicating that the
effect of the drugs on glioma (U251) cells in the chip was
significantly lower owing to the presence of the BBB.65
Despite significant progress in constructing BBB models,
there is still a long way to go to replicate the BBB invivo,
as it is a dynamic multicellular interface that regulates
the transport of molecules between blood circulation and
the brain parenchyma.94 The cell type and degree of tight
junctions in ECs are the most important factors determin-
ing the success or failure of the BBB model. They affect
the transport of related substances between the endothe-
lium and epithelium in the model because different cells
obtain different pumps, which may lead to variations in
transport and allow diverse substances to pass through.
Constituent cell types, including non- fenestrated brain
microvascular ECs, microglial cells, pericytes, astrocytes,
and neurons, play indispensable roles in BBB integrity.95
Other components, such as tight junction proteins, ad-
herens junctions, and junctional proteins, may influ-
ence barrier permeability.96 None of these compositions
can be completely simulated using a simple layer of the
epithelium.
Finally, the integration of technologies mentioned
above promotes the progress of CIVMs. Compared with
2D cultured cells, organoid techniques may improve the
performance of organ chips. A 3D proximal tubule model,
composed of epithelial cells isolated from kidney organ-
oids matured under static conditions, exhibited significant
upregulation of OCT2 and OAT1/3 transporters compared
to that of control chips based on immortalized proximal
tubule epithelial cells and was used to mimic basolateral
drug transport and uptake in drug screening and disease
modeling.97
CIVM for precision medicine
With the development of medical science, the range of
drugs and treatment options for patients and doctors has
grown. However, due to patient- specific reactions and
drug side effects, particularly in the treatment of tumors
and rare diseases, there is growing concerns regarding pre-
cision medicine that could match patients' individualized
medication needs.98 Consequently, CIVM fulfilled the de-
mand for precision medicine.
Welm et al.99 developed tumor organoids from a
43- year- old patient with triple- negative breast cancer,
screened a library of FDA- approved and experimental
drugs, and found that eribulin and talazoparib emerged
as promising candidates, whereas several of the chosen
clinical therapies did not appear to be effective. This re-
sult matched the clinical treatment result, showing that
the patient experienced early metastatic recurrence in
the liver after serial treatment. Liver metastases and as-
cites regressed completely after eribulin treatment. Wang
et al.,100 developed PDOs and found that they mirrored
patient clinical responses to platinum chemotherapy and
displayed drug response heterogeneity to targeted agents,
including PARPis. Additionally, they found that the use
of combination strategies targeting the resistance mech-
anisms of a patient who relapsed during olaparib main-
tenance therapy could reverse the effects. Besides PDOs,
including cervical cancer, head and neck squamous cell
carcinoma, prostate cancer, lung cancer, rectal cancer, and
endometrial cancer were developed as platforms to deter-
mine the effects of chemotherapy, radiation therapy, and
targeted therapy in patients.
In addition to traditional chemical and targeted drugs,
tumor immunotherapy has emerged as a new hope for
cancer treatment. Immunotherapy is used to control
and eliminate tumors by restarting and maintaining
the tumor- immune cycle and restoring anti- tumor im-
mune response.101 Thus, immunotherapy is expected to
have a higher demand for individualized drug selection.
Furthermore, normal tumor PDOs and organ chips can-
not be used to test the efficacy of tumor immunotherapy.
Therefore, CIVMs, such as tissue slices, immune co-
culture organoids, and organ chips, may be an alternative.
Dijkstra et al. expanded autologous peripheral blood
mononuclear cells (PBMCs) and established autologous
tumor organoids. PBMCs were then stimulated weekly
with tumor organoids to induce tumor- specific T- cell
responses. This co- culture system enriched the tumor-
reactive CD8+ T cells from PBMCs.102 Tumor reactivity
of the expanded autologous CD8+ T cells was analyzed.
Organoid systems can be used to evaluate the efficiency
of immune- related tumor destruction.102 Zhou etal. es-
tablished a 3D co- culture system consisting of patient-
derived cholangiocarcinoma organoids and immune cells
(such as PBMCs or purified T cells) and studied organoid
cell death caused by PBMCs and purified T cells. Distinct
responses of different PDOs to direct and indirect contact
with immune cells were noted.103 These results indicate
that CIVMs with immune cells is technologically feasible
and has great applications in assessing specific reactions
to immunotherapies invitro.
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CIVMS AND THEIR APPLICATION IN BIOMEDICAL
Based on the development of microfluidic and im-
mune co- culture techniques, CIVM- containing immune
cells have shown great potential in predicting patient
reactions to immunotherapies.104 The efficiency of ICIs
such as PD- 1 and PDL- 1 antibodies was determined
using such a model.105 Studies on high- grade serous
ovarian cancer (HGSC) have indicated that bispecific
PD- 1/PDL- 1 antibody showed superior efficiency com-
pared to monospecific anti- PD- 1 or anti- PD- L1 antibod-
ies in HGSC organoid/immune cell co- culture models.106
Primary chordoma PDOs have been generated and used
to test the dose- dependent effects of nivolumab in pre-
dicting treatment responses.107 Besides, Ou etal. gener-
ated melanoma PDOs and determined that αPD- 1 can
reinvigorate CD8+ T cells and then induce melanoma
cell death.105 Junk etal. fabricated an exvivo non- small
cell lung cancer tissue slice model that maintained the
morphological characteristics of tumor specimen for at
least 12 days and maintained T- cell function for 10 days
in vitro.108 Further, the tumor- killing effect and T cell
responses to nivolumab (a PD- 1 antibody) treatment in
this model were evaluated, and the results were com-
pared with paired clinical outcomes. The two groups of
tissue samples were successfully correlated with their
clinical outcomes. Adoptive cell transfer therapies, such
as tumor infiltrating lymphocyte transfer, chimeric an-
tigen receptor (CAR) T cell transfer, and CAR NK- cells
transfer were also determined using CIVM- containing
immune components.
DISCUSSION AND CONCLUSION
CIVM represents an alternative tissue model with biomi-
metic human pathophysiology, effectively bridging the
gap between animal studies and clinical trials. It can help
identify critical biological mechanisms as well as test drug
efficiency and toxicity in target organs at the preclinical
development stage, thus providing a reliable reference for
clinical trials in the drug development pipeline. Moreover,
CIVM- like PDOs or patient- derived organ- on- chips can
contribute to precision medicine, by evaluating the per-
sonalized sensitivity of clinical treatment, exploring
mechanisms of resistance, and identifying effective strate-
gies to address human heterogeneity.
The platform integrates different technologies, such as
invitro cell culture, organoid, co- culture, and microfluidic
techniques, and serves as a product of multidisciplinary
and interdisciplinary science. It enables the monitoring of
drug metabolism pathways and toxicity effects on target
cells/organoids, while enhancing the efficacy and reliabil-
ity of the experimental outcomes. Additionally, the com-
bination of drug sensitivity tests and gene sequencing/
analysis can facilitate appropriate and personalized treat-
ment strategies for patients.100
Despite the rapid and sustained progress in the de-
sign and construction of biomimetic CIVM, some lim-
itations that hinder its widespread application remains.
The first limitation is that CIVM, such as organoids, tis-
sue slices, and organ- on- chips do not fully recapitulate
the microenvironment and fall short in completely em-
ulating the complex physiology of organs or the human
body. As a refined experimental model, even the most
complex multi- organ chips are far from real human or-
gans and systems in terms of structures and integrated
functions. Interpretation of results obtained by CIVMs
may inadvertently overlook safety signals that could be
indicated in animal studies. This may be the key issue
limiting the use of the CIVM for preclinical evaluation
instead of animal experiments. Second, there are still
some drugs or treatments (for example, tumor immu-
notherapy and anti- angiogenic drugs) that are difficult
to predict, although numerous organoids and organs-
on- chips have been widely studied and established to
recreate patient responses to drugs. Tumor immunology
and angiogenesis are intricate, multifaceted events that
are harmonized by several organs or systems in vivo
and need precise regulations.109 Tissue slices and im-
mune co- culture might be optimal choices to reproduce
the immune microenvironment invitro. However, dis-
parities in the detection index to evaluate the model's
response to immunotherapeutic drugs remain challeng-
ing. The third limitation is the absence of standardized
and quality control criteria for materials (such as cells,
tissues, proteins, and microfluidics) used in CIVMs. Cell
culture practices vary in different laboratories, and the
vitality of primary tissues or cells varies in different pa-
tients. This causes instability in the materials used in
CIVMs. Moreover, because the formation and applica-
tion of CIVMs are still in the early research phase, de-
termining the most appropriate parameter conditions
remains challenging. In addition, these factors make it
difficult to formulate universally recognized standards
and quality control criteria, which are essential to en-
sure the reproducibility and robustness of CIVM for in-
tended applications in drug discovery and development.
Thus, CIVMs need to be designed and improved based
on experimental data.
Fortunately, novel technologies, such as cell sheet en-
gineering, 3D bio- printing, and even four- dimensional
(4D) bio- printing, are rapidly being developed as pow-
erful tools. Overall, the advancement of CIVM and
the establishment of an appropriate evaluation index
for drug assessment will underscore the suitability of
CIVMs for the evaluation of various drug characteristics
and administration modes, thereby achieving a more
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WANG etal.
comprehensive and extensive assessment of the drug or
treatment invitro.
AUTHOR CONTRIBUTIONS
L.W., D.H., and J.X. contributed equally to this work. L.W.,
resources, writing – review and editing, visualization.
D.H. resources, writing – original draft, writing– review
and editing. J.X. writing – revise manuscript writing. J.H.
conceptualization, resources, writing – review and editing,
supervision. Y.W. conceptualization, resources, writing –
review and editing, visualization, supervision. All authors
have read and agreed to the final manuscript.
FUNDING INFORMATION
This study was supported by The National Key Research
and Development Program of China (2022YFC2407303);
Major Science and Technology Projects of Zhejiang
Province (2020C03058); Research Center for Lung
Tumor Diagnosis and Treatment of Zhejiang Province
(JBZX- 202007).
CONFLICT OF INTEREST STATEMENT
The authors declared no competing interests for this
work. D.H. is a lab researcher of Hangzhou Chexmed
Technology Co., LTD. Y.W. is chief executive officer of
Hangzhou Chexmed Technology Co., LTD.
ORCID
Luming Wang https://orcid.org/0009-0001-3020-4210
Danping Hu https://orcid.org/0000-0002-8563-0444
Jinming Xu https://orcid.org/0009-0001-4316-9661
Jian Hu https://orcid.org/0000-0002-9494-9828
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How to cite this article: Wang L, Hu D, Xu J, Hu
J, Wang Y. Complex invitro model: A
transformative model in drug development and
precision medicine. Clin Transl Sci. 2024;17:e13695.
doi:10.1111/cts.13695
