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Biomimetic cell culture for cell adhesive propagation for tissue engineering strategies

October 2023
Materials Horizons 10(11)

October 2023
10(11)

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Biomimetic cell culture, which involves creating a biomimetic microenvironment for cells in vitro by engineering approaches, has aroused increasing interest given that it maintains the normal cellular phenotype, genotype and functions displayed in vivo. Therefore, it can provide a more precise platform for disease modelling, drug development and regenerative medicine than the conventional plate cell culture. In this review, initially, we discuss the principle of biomimetic cell culture in terms of the spatial microenvironment, chemical microenvironment, and physical microenvironment. Then, the main strategies of biomimetic cell culture and their state-of-the-art progress are summarized. To create a biomimetic microenvironment for cells, a variety of strategies has been developed, ranging from conventional scaffold strategies, such as macroscopic scaffolds, microcarriers, and microgels, to emerging scaffold-free strategies, such as spheroids, organoids, and assembloids, to simulate the native cellular microenvironment. Recently, 3D bioprinting and microfluidic chip technology have been applied as integrative platforms to obtain more complex biomimetic structures. Finally, the challenges in this area are discussed and future directions are discussed to shed some light on the community.

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Materials

Horizons

rsc.li/materials-horizons

REVIEW ARTICLE

Zhaoying Wu, Lin Xiao et al .

Biomimetic cell culture for cell adhesive propagation for

tissue engineering strategies

ISSN 2051-6347

Volume 10

Number 11

November 2023

Pages 4637–5316

4662 | Mater. Horiz., 2023, 10, 4662–4685 This journal is © The Royal Society of Chemistry 2023

Cite this: Mater. Horiz., 2023,

10, 4662

Biomimetic cell culture for cell adhesive

propagation for tissue engineering strategies

Qiuchen Luo,

Keyuan Shang,

Jing Zhu,

Zhaoying Wu, *

Tiefeng Cao,

Abeer Ahmed Qaed Ahmed,

Chixiang Huang

and Lin Xiao *

Biomimetic cell culture, which involves creating a biomimetic microenvironment for cells in vitro by

engineering approaches, has aroused increasing interest given that it maintains the normal cellular

phenotype, genotype and functions displayed in vivo. Therefore, it can provide a more precise platform

for disease modelling, drug development and regenerative medicine than the conventional plate cell

culture. In this review, initially, we discuss the principle of biomimetic cell culture in terms of the spatial

microenvironment, chemical microenvironment, and physical microenvironment. Then, the main

strategies of biomimetic cell culture and their state-of-the-art progress are summarized. To create a

biomimetic microenvironment for cells, a variety of strategies has been developed, ranging from

conventional scaﬀold strategies, such as macroscopic scaﬀolds, microcarriers, and microgels, to

emerging scaﬀold-free strategies, such as spheroids, organoids, and assembloids, to simulate the native

cellular microenvironment. Recently, 3D bioprinting and microfluidic chip technology have been applied

as integrative platforms to obtain more complex biomimetic structures. Finally, the challenges in this

area are discussed and future directions are discussed to shed some light on the community.

Wider impact

Biomimetic cell culture, which is committed to creating a biomimetic microenvironment for cells in vitro by engineering approaches, has aroused increasing

interest. In this review, initially we discuss the principle of biomimetic cell culture in terms of the spatial, chemical, and physical microenvironments. Then, the

main strategies of biomimetic cell culture and their state-of-the-art progress are summarized. To create a biomimetic microenvironment for cells, a variety of

strategies has been developed ranging from conventional scaﬀold strategies, such as macroscopic scaﬀolds, microcarriers, and microgels, to emerging scaﬀold-

free strategies, such as spheroids, organoids, and assembloids, to simulate the native cellular microenvironment. Recently, 3D bioprinting and microfluidic

chip technology have been applied as integrative platforms to obtain more complex biomimetic structures. Finally, the challenges in this area are discussed,

and future directions outlooked. To the best of our knowledge, this is the first review discussing material design and manufacture from the perspective of

biomimetic cell culture, which is a new concept or new way of thinking. It will shed some light for the materials science community, particularly concerning

biological, therapeutic and tissue engineering materials.

1. Introduction

Biomimetic cell culture involves creating a biomimetic micro-

environment for cells by engineering approaches in vitro, where

cells can grow, proliferate, diﬀerentiate, communicate, and

maintain their metabolic activities and functions as in vivo.

Thus, biomimetic culture of cells has great potential in

applications such as disease modelling, drug development

and screening, and stem cell research.

Initially, cell cultures

are performed on two-dimensional (2D) planes,

such as Petri

dishes and porous plates, on which cells are grown against

the wall. However, traditional cell 2D culture methods cannot

simulate the in vivo growth environment of cells, leading to a

distorted cellular morphology and function and limitations in

the formation of tissue-specific structures.

1,3

In addition, live animal experiments are often required after

in vitro studies with 2D cultured cells. Living animal models

can provide in vivo conditions, which are deemed more reliable

than 2D cell culture systems. However, animal experiments are

also limited due to intrinsic species diﬀerences, ethical issues,

and high costs. Recently, it was reported that animal tests are

School of Biomedical Engineering, Shenzhen Campus of Sun Yat-Sen University,

Shenzhen 518107, China. E-mail: xiaolin23@mail.sysu.edu.cn,

wuzhy37@mail.sysu.edu.cn

Department of Gynaecology, First Aﬃliated Hospital of Sun Yat-Sen University,

Guangzhou 510070, China

Department of Molecular Medicine, Biochemistry Unit, University of Pavia,

27100 Pavia, Italy

Received 3rd June 2023,

Accepted 29th August 2023

DOI: 10.1039/d3mh00849e

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no longer required before human drug trials by the FDA.

This

calls for more advanced and precise in vitro models, in which

achieving biomimetic cell culture is a fundamental issue.

Accordingly, tissue and organ engineering based on biomi-

metic cell culture may provide reliable in vitro models.

2. The principle of biomimetic cell

culture

The extracellular matrix (ECM) is a non-cellular three-

dimensional (3D) macromolecular network outside of cells.

It provides structural support, regulates and facilitates cell–cell

and cell–tissue signaling and controls the morphology, structure

and function of cells.

The three main components of the ECM

are fibrin, glycoproteins and proteoglycans, which regulate the

biochemical and mechanical properties, provide sufficient cell–

ECM interactions and mediate cell–cell interactions.

6,7

Therefore,

the key to achieving biomimetic cell culture is to recapitulate the

ECM in vitro to provide a simulated growth environment for cells

as in vivo.

In general, the cellular microenvironment in vivo can be

approximately divided into three aspects, i.e.,thespatialmicro-

environment, chemical microenvironment, and physical microen-

vironment (Fig. 1). The spatial microenvironment refers to the 3D

spatial niches where cells live in terms of the spatial geometry,

composition and spatial distribution of cells and ECM. In addi-

tion, cells in vivo grow in a 3D space with multiple chemical

signals, including growth factors, nutritional factors and signaling

factors, and various physical signals, such as mechanical cues of

the ECM.

Therefore, the basic principle of biomimetic cell culture

lies in the simulation of the spatial, chemical, and physical

microenvironments in vitro for cells.

2.1 Spatial microenvironment simulation

The 3D spatial microenvironment is crucial for cells to maintain

their normal morphology, metabolism, function, and communi-

cation.

In terms of spatial geometry, the construction of porous

scaﬀolds is a regular approach to simulate the spatial charac-

teristics of cells for biomimetic culture. The 3D spatial structures

of porous scaﬀolds, including porosity, pore size and orientation,

have a remarkable impact on the fate of cells. Porous materials

with a high specific surface area will increase the adsorption of

proteins, thus promoting cell adhesion.

In addition, a porous

surface improves the mechanical interlock between the implant

biomaterials and surrounding tissues, such as natural bone,

providing greater mechanical stability at this critical interface.

Studies have shown that a porosity of more than 50% and an

interconnected pore size of more than 100 mm are the minimum

requirements for cell growth and migration.

He et al. prepared

a porous scaﬀold with a pore size of ca. 167 mm, which was

suitable for osteoblasts to infiltrate the scaﬀold and facilitate the

transport of nutrients and metabolic waste.

In bone tissue

engineering, biomaterials with smaller pore sizes are beneficial

for the formation of anoxic conditions and induce osteochon-

drogenesis before osteogenesis, while biomaterials with larger

pore sizes lead to vascularization and direct osteogenesis with-

out forming previous cartilage.

In addition, the orientation

of pores is closely related to the direction of cell migration. For

example, Jiang et al. prepared a radial porous nanocomposite

chitosan (CS)/HA scaﬀold using HA nanoparticles from porcine

cortical bone and CS by freeze-casting technology (Fig. 2a).

The

scaﬀold exhibited excellent biological functions and enabled

cells to attach and migrate along the pore channels in vitro.In

addition, Zhang et al. reported that oriented pores are more

conducive to cell ingrowth and interaction and more eﬃcient

nutrient transport and ECM deposition than scaﬀolds with a

random orientation.

The spatial microenvironment also involves the interaction

between cells and the ECM. The components and microstruc-

ture of the ECM provide an elaborate 3D niche for cells to carry

out physiological activities and largely aﬀect cell behaviors.

For example, collagen and its derivatives are widely applied as

biomimetic ECM in cartilage tissue engineering.

In addition,

the distribution of cells also features the spatial microenviron-

ment. This may only be a single layer of cells with cell–cell

interactions in the horizontal direction in conventional 2D cell

culture, while the distribution of cells is multilayered with

all-directional cell–cell interactions in biomimetic cell culture.

For example, in cellular spheroids, the distribution of cells is

compact with all-directional cell–cell interactions, which is

conducive to maintaining cell activity and function.

In addition, the spatial microenvironment in vivo is dynamic

in response to various chemical and physical cues. To achieve a

dynamic spatial microenvironment, intelligent constructs

whose properties change over time based on 3D cell culture

systems were developed, also known as four-dimensional (4D)

cell culture.

They respond to diﬀerent stimuli, including

electric field, magnetic field, temperature, humidity, pH and

light.

21,22

Miao et al. prepared an intelligent 4D scaﬀold with

shape-memory characteristics (Fig. 2b).

Its topography can be

changed dynamically from microwell arrays to aligned patterns

over time by changing the environmental temperature. The

results showed that the microwell arrays promoted the for-

mation of neural stem cell (NSC) aggregates and early diﬀer-

entiation, while the aligned patterns were conducive to neurite

extension and elongation.

Some researchers also designed

Fig. 1 Typical schematic of the cellular microenvironment.

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chemically modular microgels for logical degradation to achieve

customizable void ratios in 3D-printed scaﬀolds at specific

times.

2.2 Chemical microenvironment simulation

Cells are in a microenvironment with a large number of

biochemical factors. Due to the presence of these factors, cells

can normally grow, proliferate and perform a series of physio-

logical functions. For example, mesenchymal stem cells (MSCs)

require a series of growth factors, including transforming

growth factor-b(TGF-b), hepatocyte growth factor (HGF),

interleukin-1 (IL-1) and interleukin-6 (IL-6),

to achieve func-

tions such as fixation, migration, proliferation, and diﬀerentia-

tion. Additionally, the communication between cells includes

bidirectional transmission of various biochemical factors. Also,

the chemical signals in the intracellular environment play a

certain role in regulating the growth and diﬀerentiation of cells.

Therefore, including these chemical signals is an important

aspect of biomimetic cell culture. For example, Li et al.

constructed scaﬀolds that can release TGF-b3 in an orderly

manner, which is conducive to the migration, proliferation,

and fibrocartilage diﬀerentiation of cells in vitro in the culture

system, and also remodelling the fibrous matrix into a fibrous

cartilage matrix.

In another study, Lee et al. developed a dual

cryogel system consisting of a gelatin/CS cryogel surrounded by

a gelatin/heparin cryogel for the dual delivery of vascular

endothelial growth factor (VEGF) and bone morphogenetic

protein-4 (BMP-4) (Fig. 2c).

The release of these two factors

enhances the osteogenic diﬀerentiation of adipose-derived

stem cells (ADSCs).

The chemical microenvironment of cells in vivo also includes

the biochemical properties of ECM components, which have

remarkable impacts on cell adhesion, growth, proliferation, and

migration. Based on this, the biochemical properties of bio-

materials in contact with cells are also closely related to the

growth of cells in vitro. For instance, dopamine (DA) was

partially covalently grafted onto a hyaluronic acid–chondroitin

sulfate composite gel to enhance the matrix stability in the cell

culture medium. DA, as a neurotransmitter, plays a critical role

in axonal outgrowth and stimulates the remodelling properties

of gels by capturing cell-secreted laminin and binding brain-

derived neurotrophic factor.

This study implemented the

interaction between cells and biological scaffold materials,

and ultimately successfully simulated the formation of neuro-

nal networks. Additionally, RGD peptides are often used to

modify biomaterials to increase their cell adhesion ability.

Fig. 2 (a) Schematic diagram of the structure and application of nanocomposite CS/HA scaﬀolds. Radial porous scaﬀolds can cause cells to go deep

into the scaﬀolds along the pore channel (curved arrows), while preventing unrelated cells from invading the scaﬀold. Axial porous scaﬀolds allow cells to

migrate from the bottom pore channel (curved arrows) and allow fibroblasts to invade the scaﬀolds. Reproduced with permission.

Wiley-VCH. (b) Schematic diagram of the preparation of new intelligent 4D scaﬀolds. (I) Fused deposition modelling (FDM) printing molds. (II) Coating

molds with polydimethylsiloxane (PDMS) to solidify to obtain PDMS microgrooves. (III) Extruding 4D printed materials into PDMS microgrooves for

solidification. (IV) Imprinted polymethyl methacrylate microwell arrays by stereolithography (STL). (V) Imprinted 4D scaﬀolds from microwell molds at

60 1C for 10 min. (VI) Microwell array fabrication. (VII) Cell seeding scaﬀolds. (VIII) Time-dependent scaﬀold materials. Reproduced with permission.

(d) Photosensitive hypertensive metamaterial scaﬀolds for dynamic cell culture systems. (i) Dynamic culture system consists of an elastic styrene–

ethylene–butylene–styrene framework and photoactive surface, allowing the response to light and mechanical stimulation. (ii) Dynamic regulationof

cells by dynamic culture systems under different external stimulations. Reproduced with permission.

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In addition, to achieve a biomimetic culture of cells in vitro,

it is also necessary to have an adequate nutrient supply

and meet the growth conditions of cells, such as an appro-

priate oxygen concentration and soluble nutrient molecule

concentration.

2.3 Physical microenvironment simulation

The cellular microenvironment involves various physical signals

mediated by the ECM and interstitial fluid, which in turn aﬀect

the fate of cells. These physical signals include not only mechan-

ical eﬀects produced by cell–cell interactions but also mechanical

cues produced by physical factors such as the morphological

structure of materials, mechanical properties of the ECM, internal

stress, and external forces in the cellular microenvironment.

Essential physical signals, such as the appropriate mechan-

ical stimulation and mechanical properties of materials, are

crucial in the biomimetic culture of cells.

Zhang et al. applied

cyclic compression to MSCs, inducing them to produce func-

tional osteocyte bone organoids.

It was found that cyclic

pressure enhanced the diﬀerentiation of osteoblasts and orga-

noid mineral density and stiﬀness. Nguyen et al. used aldehyde

hyaluronic acid to oxidize gelatin microcarriers to produce a

smoother morphology and stronger stiﬀness.

The resultant

microcarriers had a higher aﬃnity for corneal stromal cells and

were conducive to their attachment and growth.

Mechanical signals from internal stress and external stimu-

lation are essential for many cells and tissues in vivo, such as

chondrocytes and articular cartilage. Physical signals generated

by proper mechanical loading (such as exercise) can induce

homeostatic regulation in vivo to achieve reorganization and

maintenance of the cartilage ECM.

This inspired researchers

to optimize the in vitro culture of chondrocytes (or precursor

cells) and cartilage tissue engineering by introducing appro-

priate mechanical stimulation. It has been verified that

mechanical stimulation can trigger several responses in vitro,

such as increased secretion of osteopontin and other ECM

proteins.

For example, Paggi et al. applied multidirectional

compression to hydrogels containing chondrocytes by a

cartilage-on-chip device.

It was found that the glycosamino-

glycans secreted by the cells significantly increased after this

mechanical stimulation. Additionally, the expression of specific

cartilage ECM markers, such as aggrecan, collagen II, and

collagen VI, increased. Table 1 presents a summary of the

typical mechanical stimulation compatible with diﬀerent cells.

In addition to mechanical signals, other physical signals,

such as light and electrical stimulation, have also been applied

to cell culture in vitro. Chen et al. constructed a dynamic cell

culture system capable of responding to mechanical and light

stimulation (Fig. 2d).

The flexibility and tensile elasticity of

the dynamic culture system enabled it to serve as a dynamic

matrix for activating cell activity. Light stimulation was applied

to produce mild heat stress, which together with mechanical

stimulation could eﬀectively improve the activity of stem cells

and their ability to diﬀerentiate into cardiomyocytes. In

another study, appropriate electrical stimulation was used to

control the cell morphology and phenotype in biomimetic

cultures of human-induced stem cell-derived cardiomyocytes

(hPSC-CMs).

Electrical stimulation was also employed in the

biomimetic culture of chondrocytes, which has broad poten-

tial for exploring mechanotransduction in cartilage tissues.

Recently, researchers developed electroactive/conducting

hydrogels and scaﬀolds.

Electroactive materials can not only

promote cell activity but also simulate the electrophysiological

microenvironment of natural electrically excitable cells through

their own electroactive properties and transmit biochemical

signals.

46,47

For example, Li et al. prepared electroactive fibrous

scaﬀolds by incorporating barium titanate nanoparticles. The

dielectric permittivity of the scaﬀolds is comparable to that of

natural bone, which enhanced the osteogenic diﬀerentiation of

MSCs.

Malki et al. reported the preparation of an electroactive

Table 1 Application of mechanical stimulation in biomimetic cell culture

Cell type Mechanical stimulation Eﬀectiveness Application Ref.

Preadipocyte 930 kPa culture substrate rigidity; 12% static

stretch

Higher cell metabolism and mitochondrial

activity

Acceleration of adipocyte

diﬀerentiation

Osteocyte-like

mouse cell

Unidirectional shear stress of 0.03 Pa Enhancement of cancer cell invasion with

mechanical stimulation

Construction of in vitro

prediction model of bone

metastasis

MSC Cyclic hydrostatic pressure with 10–300 kPa

pressure and 2 Hz frequency

Promotion of collagen synthesis and

mineral deposition

Promotion of osteogenic

diﬀerentiation

0.07 N preload, 1% strain, 5 Hz, 5 min per

loading time, 5 times per week

Enhancement of organoid mineral density,

stiﬀness, and osteoblast diﬀerentiation

Construction of functional

osteocyte bone organoids

Nucleus pulpo-

sus derived

stem cell

Hypoxic environment; chamber pressure

1.0 MPa

Exhibition of stronger resistance to

overloading

Treatment of disc degen-

eration and low back pain

Chondrocyte Cyclic tensile strain at 0.33 Hz, 0–10% strain Inhibition of inflammatory response Research of signal pathway 40

compression and shear strain (300 mbar

positive pressure, 350 mbar negative

pressure, 0.33 Hz)

Promotion of the expression of glycosami-

noglycans and specific cartilage ECM

markers

Cartilage tissues basic

research and drug testing

Mammary

tumor cell

1 N force at 5 Hz for 3 min Inhibition of osteoclastogenesis and the

levels of tumorigenic genes; promotion of

osteogenesis

Reduction of breast cancer-

associated bone metastasis

Heart-derived

ventricular cell

2% elongation (strain) at 0.25 Hz Reduction of apoptosis and inflammatory

factors

A new target for the

treatment of heart disease

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hydrogel of chondroitin sulphate containing stromal cell-

derived factor 1 (SDF-1). The release of SDF-1 could be con-

trolled by externally applied electrical stimulation to promote

cell migration and infiltration.

3. The main strategies of biomimetic

cell culture

Many strategies have been developed to create a biomimetic

microenvironment for cells in vitro. Conventional scaﬀolds can

provide support and create a spatial microenvironment,

chemical microenvironment, and physical microenvironment

for cells to some extent.

However, the cellular microenviron-

ment constructed by this method is relatively simple, with

limited capacities for cell arrangement and nutrient

supplementation.

Scaﬀold-free strategies, such as spheroids,

organoids, and assembloids, have enriched the cell types and

arrangements and enabled the formation of complex cellular

aggregate structures through the self-assembly of cells, which

mimic the process of natural tissue and organ formation.

Recently, fast-growing 3D bioprinting and microfluidic chip

technology provided potent platforms for integrating emerging

biomimetic culture strategies, creating more complicated and

precise biomimetic constructs.

Beyond the regular use of

individual cells, cellular aggregates such as spheroids, orga-

noids and assembloids can be applied in 3D bioprinting and

microfluidic chips.

3.1 Conventional scaﬀold strategies

The simplest way to realize biomimetic culture in vitro is to seed

cells on scaﬀolds. In this case, a variety of materials (natural or

synthetic) in diﬀerent states (gel or solid) can be used as

scaﬀolds. Porous scaﬀolds with appropriate porosities and pore

sizes can provide an ideal 3D space for cell attachment

and migration. With specific physical and chemical signals by

surface modification, mechanical adjustment or chemical/

biological functionalization of the scaﬀolds, the cells in the

scaﬀolds can grow, proliferate, and diﬀerentiate to form tissue-

like structures.

3.1.1 Solid scaﬀolds. Metals, ceramics, polymers and other

materials have been used to fabricate solid scaﬀolds for bio-

mimetic cell culture.

Metals such as titanium (Ti)

55,56

and

magnesium (Mg)

and ceramics such as hydroxyapatite (HA),

bioactive glass and b-tricalcium phosphate (b-TCP) are com-

monly used for porous scaﬀolds. As an example, Zhang et al.

developed a biodegradable, bioactive, and elastic calcium

phosphide (CaP) ceramic sponge with high porosity (approxi-

mately 99%).

It was self-assembled by HA nanowires and

b-TCP nanofibers into a seamless interwoven network, which

was conducive for cell attachment, proliferation, and osteogenic

diﬀerentiation. Recently, many techniques, such as non-solvent-

induced phase separation, thermally induced phase separation,

foaming, electrospinning, magnetic levitation, self-assembly,

extrusion-based cell entrapment and bioprinting, have been

developed for the fabrication of scaﬀolds.

The topological

structure (porosity and pore size), chemical (biocompatibility

and degradability) and physical (mechanical and electroactive

properties) characteristics of the materials are essential for the

construction of the spatial, chemical and physical microenviron-

ment. The porous structure and mechanical properties of scaf-

folds can aﬀect the morphology, adhesion, growth, maturation,

and metabolism of cells.

60,61

However, there are some limitations

in the solid scaﬀold approach, especially when using large scaf-

folds. For example, cells cannot migrate into the inner pores of

scaﬀolds and only adhere to the shallow surface. Even if there are

cells in the inner pores, their activity will be reduced due

to insuﬃcient nutrient supplementation and signal exchange.

Moreover, a suitable degradation rate of scaﬀold materials that

matches the cell recovery rate is required for most applications of

scaﬀold materials. It is expected that with the degradation of

scaﬀold materials, cells undergo proliferation and diﬀerentiation

and form tissue structures with biological functions. However,

this is currently a major challenge in the development of scaﬀold

materials. This has caused numerous failures in scaﬀold material

applications due to the unmatchable material degradation and

cell recovery. The rapid degradation of scaﬀold materials may lead

to inadequate cell development and poor treatment eﬃcacy, while

delayed material degradation may cause inflammation.

Addi-

tionally, the degradation products of the scaﬀold materials should

be non-cytotoxic and cause minimal fibrosis and foreign body

reactions, which further increase the diﬃculties in material

development.

Compared with macroscopic solid scaﬀolds, microcarriers

based on porous microspheres have significant advantages in

cell loading capacity and nutrient supplementation because of

their microscopic size and high surface area to mass ratio.

64–66

Microcarriers are micron-level porous systems, with a particle

size in the range of 100–300 mm.

Microcarriers with appro-

priate porous structures allow cells to attach, grow, and migrate

within the internal areas and facilitate the diffusion of nutri-

ents, oxygen and metabolites. For example, Huang et al. pre-

pared porous chitosan microcarriers with interconnected pores

of a suitable size using a method of emulsion-based thermally

induced phase separation. By using these microcarriers, 3D

culture of L-02 hepatocytes was achieved, where cells grew and

migrated in the internal pores and formed multidirectional

cell–cell interactions.

In addition, microcarriers can protect

cells from shear damage when incubated in an agitating

suspension in bioreactors.

3.1.2 Hydrogels. Hydrogels are a type of 3D network

formed by embedding hydrophilic polymer chains in a water-

rich environment.

Hydrogels are promising materials for

biomimetic cell culture because of their porous network struc-

ture, high water content (470% water) and good biocompat-

ibility, which are similar to the natural ECM.

For example, a

curdlan/polyvinyl alcohol composite hydrogel had an appropri-

ate pore size and porosity to simulate the spatial microenviron-

ment and could maintain cell activity when mouse fibroblasts

were cultured inside.

Hydrogels have tailorable viscoelastic

and stiﬀness properties depending on their composition

and crosslinking mechanisms, including physical crosslinking

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(ionic, H-bonding, or hydrophobic) and chemical crosslinking

(covalent bonds between macromolecular chains).

72–74

The

viscoelastic nature of hydrogels endow them with the ability

to resist mechanical deformation. In particular, dynamic cross-

linked hydrogels will relax and deform under stress to promote

cell diffusion, proliferation and differentiation.

75,76

In addition,

the structure of hydrogels can include a variety of biochemical

signal molecules and allow their diffusion and release in a

preset manner, which simulates the biochemical features of

the cellular microenvironment. Hydrogels can also deliver

external physical stimuli to cells to further shape the cellular

microenvironment.

However, similar to solid scaﬀolds, large hydrogels also have

limitations in cell arrangement and nutrient supplementation.

Alternatively, microgels have attracted increasing attention

for biomimetic cell culture.

The surface wettability, hydro-

phobicity, charge, and chemical properties of microgels

can aﬀect cell attachment, proliferation, migration, and self-

organization.

78,79

Microgels are also used to fabricate macro-

porous hydrogels by assembly. Rommel et al. reported a 3D

soft macroporous hydrogel scaﬀold by the assembly of two-

component rod-like microgels with a defined size, stiﬀness,

and reactivity.

In this system, rod-like microgels act as

artificial ‘‘blood vessels’’ to deliver oxygen and nutrients, and

thus cells in both the internal and external areas of the

hydrogels can obtain suﬃcient oxygen and nutrients.

3.2 Scaﬀold-free strategies

Cellular aggregates such as spheroids, organoids and assem-

bloids have been developed as next-generation strategies of

biomimetic cell culture, in which cells are self-organized into

aggregates without a scaﬀold support. A variety of methods can

be used to promote the formation of cellular aggregates, such

as using hanging drop microplates and low adhesion plates

with ultralow attachment coatings.

Because of the native-like

microtissue structures with abundant cell–cell and cell–ECM

interactions, the cells in these aggregates have a microenviron-

ment closer to that of cells in vivo than cells cultured in

scaffolds.

3.2.1 Spheroids. Spheroids are simple clusters of multiple

cell types, such as tumor spheroids,

neurospheroids,

hepa-

tocyte spheroids

and stem cell spheroids.

Compared with

2D cell lines, cells in spheroids are much closer to cells in vivo

in terms of cellular morphology, metabolic activity, and

function.

In addition to their 3D spatial structure, spheroids

can form a biomimetic chemical microenvironment with

gradients of nutrients, gases, growth factors and signaling

factors.

Moreover, mechanical stimulation, such as compres-

sion force, shear force, hydrostatic pressure and osmotic pres-

sure, can be applied to spheroids by adjusting the culture

environment.

Spheroids are formed because adherent cells tend to aggre-

gate under low attachment conditions. The main mechanism of

spheroid formation is that cells self-organize to establish cell–

cell contacts in response to external physical stimulation, such

as gravity, magnetic force, and sound waves. For example,

Byun et al. used magnetic force to self-assemble cells to form

spheroids of ADSCs (Fig. 3a).

They prepared a magnetized

artificial ECM containing magnetic nanoparticles (MNPs). The

artificial ECM had a stable spatial structure, and ADSCs were

assembled into composite magnetized spheroids, which were

used as building blocks for further 3D tissue engineering. The

ADSC spheroids showed a crucial influence on cell proliferation

and differentiation, and they successfully induced chondrocyte

differentiation. Rasouli and Tabrizian reported a fast spheroid

formation technique based on boundary-driven acoustic waves

to produce dense cellular aggregates (Fig. 3b).

In this study,

when solutions were introduced into microfluidic channels and

reached the acoustic region, cells were condensed and com-

pacted into spheroid clusters within seconds by acoustically

induced hydrodynamics. Using this method, biomimetic cocul-

ture spheroids could be obtained from malignant breast tumor

cells (MDA-MB-231) and human breast adenocarcinoma cells

(MCF-7).

Moreover, the eﬀects of chemical signals can also cause cells

to form spheroids. Because the hydrated surface of hydrogels

can inhibit the adsorption of proteins and mediate the inter-

action force between cells, cells will not adhere to the surface of

Fig. 3 (a) Schematic of the construction of composite stem cell spher-

oids. They are constructed from synthetic fibres (MSFs) and complex tissue

structures (rings and laminates) by magnetic assembly. Because MSFs

hinder cell contraction, cell spheroids still exhibit structural stability over

7 days of culture. Reproduced with permission.

Publishing. (b) Formation mechanism of acoustic spheroids assembled by

collagen. Cells surrounded by collagen fibres are injected into microfluidic

channels. Driven by sound waves, cells are captured and remodelled into

spheroids. Reproduced with permission.

permission.

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the hydrogel material, which results in the formation of spher-

oids. Ai et al. developed a supramolecular polypeptide hydrogel

via the self-assembly of biotinylated peptides for biomimetic

cell culture (Fig. 3c).

The introduction of a D-amino acid into

the polypeptide may reduce the cell–ECM interaction, and thus

promote the spontaneous formation of spheroids. In another

study, thermo-responsive hydrogels with surface micropore

patterns were developed to culture ADSC spheroids and deliver

them into Matrigel to study their migration and fusion

behaviors.

In addition, Im et al. investigated the effect of

seeding density on the formation of spheroids.

Studies have

found that the expression levels of angiogenic paracrine factors

and the secretion of cell adhesion molecules in spheroids are

different with various seeding densities of human ADSCs.

Under appropriate seeding density conditions, the spheroids

showed significantly enhanced therapeutic angiogenic capa-

city. Through the above-mentioned physical and chemical

methods, it is possible to control the number of cells and the

size of the spheroids, to produce high-density cell clusters and

increase the ratio of cells to fluid.

3.2.2 Organoids. More than 60 experts have accurately

defined organoids as three-dimensional structures derived

from (pluripotent) stem cells, progenitors, and/or diﬀerentiated

cells that self-organize through cell–cell and cell–matrix

interactions to recapitulate aspects of the native tissue archi-

tecture and function in vitro.

Organoid technology is one of

the most potent approaches for biomimetic cell culture. Orga-

noids have organ specificity in many aspects, such as cell type,

function, and spatial structure.

The cell source, culture

method and ECM material are three decisive factors in the

culture of organoids. Organoid formation requires cells to be

embedded in an appropriate ECM and supplemented with an

appropriate organic medium and a range of growth factors to

promote the self-organization and growth of the cells

(Fig. 4a).

With specific growth factors, stem cells proliferate,

differentiate and self-organize into organ-like structures, which

can be passaged and cultured and exhibit a high degree of

genetic stability.

A variety of stem cells, including embryonic

stem cells (ESCs), adult stem cells (ASCs) and induced plur-

ipotent stem cells (iPSCs), has been used for the formation of

organoids. To induce the specific development of organoids,

the culture medium is usually supplemented with several

ligands or compounds, such as retinoic acid, BMP, and epider-

mal growth factor (EGF), which can activate pivotal modal

signaling pathways.

The physical conditions of the ECM

environment significantly influence the morphology of the

self-organized organoids (Fig. 4b). During the initial stages of

organoid development, the suspension culture conditions

Fig. 4 (a) Schematic of organoid formation and self-organization: diverse types of cells (diﬀerent colors) sort themselves to form the embryo body over

spatial constraints, eventually generating more diﬀerentiated cells and developing organoids. Reproduced with permission.

VCH. (b) Methods to induce organoid formation: suspension culture, bioreactor and ECM. Reproduced with permission.

(c) Methods for the preparation of the 3D structure of human islet organoids. (i) Methods for the construction of the islet organoid 3D structure by cell

self-aggregation. (ii) Mixed hydrogels with human islet lineage cells to generate 3D structures vi a 3D bioprinting or biomaterial embedding. (iii) Methods

to maintain islet organoids, including implantation in 3D printing scaﬀolds, decellularized pancreatic scaﬀolds and organs-on-a-chip. Reproduced with

permission.

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facilitate the reaggregation and self-sorting of the cells. After

the cells aggregate into a 3D structure, the use of bioreactors

can improve the perfusion levels of nutrient molecules and

oxygen, prolong the organoid culture time and increase the size

of organoids. In addition, the use of naturally derived ECM

facilitates the real simulation of the cellular environment and

produces organoids closer to the actual situation.

100

The methods used for the construction of human organoids

include traditional self-aggregation (such as the use of ultralow

attachment plates, spinner flasks, and biomaterial-coated

plates), modified self-aggregation (such as suspended droplets

and microporous platforms), and hydrogel embedding,

as well

as emerging methods such as 3D bioprinting, decellularized

organ scaﬀolds, and organ-on-a-chip (Fig. 4c).

Using these

methods, many types of organoids have been developed and

applied in human healthcare areas. Table 2 presents a sum-

mary of the representative organoids reported in the last few

years. For instance, Yoshihara et al. used Matrigel to culture islet

organoids in vitro and maintain their physiological functions,

which can be used to explore insulin-dependent diabetes

therapies.

101

In the generation of liver organoids, the selection

of source cells, the type and concentration of growth factors, and

thegrowthprocessoforganoidshavebeenextensivelyexplored

(Fig. 5a). During the construction of human liver organoids, the

activities of cyclic adenosine monophosphate (cAMP) and TGF-b

need to be strictly regulated to successfully expand the cells.

102

Table 2 Representative organoids developed in recent years

Type Source Culture method Results Ref.

Endoderm-

derived

organoid

Liver

organoid

Human fetal liver pro-

genitor cells

Culture in scaﬀolds made from

liver ECM

Formation of hepatocytes and bile duct

structures at the same time

106

Pluripotent stem cells

(PSCs)

Coculture in plate Long-term expansion with competent liver

functionality

107

Hepatobiliary

tubular

organoid

Hepatocytes,

cholangiocytes

Coculture in plate Reproduction of hepatocyte metabolite

transport in liver tissue

108

Hepatobiliary

organoid

iPSCs, ESCs Coculture on Matrigel-coated

plates

Demonstration of hepatic and biliary

functional attributes

109

Lung

organoid

ESCs Culture on poly (lactide-co-

glycolide) or polycaprolactone

(PCL) scaﬀolds

Formation of tubular structures similar to

adult airways

110

Bronchial epithelial cells,

lung fibroblasts, smooth

muscle cells

Triple culture in stiﬀer matrix Formation of airway simulated tubular

morphology

111

Intestinal

organoid

Small intestinal mucosa

cells

Coculture in plate Organoids containing enterocytes, goblet cells,

Paneth cells and entero-endocrine cells

112

Cells from human ileum

and duodenum

Coculture in plate, CRISPR

engineering

Demonstration of widespread germination

and nourish all sorts of cells

113

Mesoderm-

derived

organoid

Kidney

organoid

PSCs Coculture in plate and modula-

tion of Wnt

Vascularized and segmentally patterned

organoids

114

PSCs Extrusion-based cellular

bioprinting

Increasement of throughput, reduction of

organoid size and demonstration of improved

maturation and nephron number

115

PSCs Culture in kidney decellularized

ECM hydrogel

Demonstration of extensive vascular network

and endothelial cells

116

Breast

organoid

Breast epithelial cells

and breast cancer cells

Culture in 3D bioprinting ECM

hydrogel

Production of organoids/tumoroids in a

tissue-specific matrix

117

Trabecular

bone

organoid

Bone marrow mono-

nuclear cells and primary

murine osteoblasts

Coculture on thin slices of

demineralized cortical bone

Reproduction of the bone remodelling cycle 118

Cartilage

organoid

PSCs Suspension culture Successfully bridge of critical size long bone

defects

119

Bone callus

organoid

MSCs Digital light processing (DLP)

printing of cell-loaded hydrogel

microspheres

Maturation and formation of bone-like tissue

in vivo

120

Woven bone

organoid

MSCs 3D self-organizing coculture of

osteoblasts and osteocytes

Formation of network and communicate by

expression of sclerostin

121

Bone marrow

organoid

MSCs Coculture in transglutaminase-

polyethylene glycol/hyaluronic

acid hybrid hydrogels

Functional bone marrow organoids with a

higher level of complexity

122

Ectoderm-

derived

organoid

Sweat gland

organoid

Reprogram human epi-

dermal keratinocytes

Coculture in plate Self-organization of into monoclonal spheres

and organoids

123

Skin organoid iPSCs Coculture in plate Production of hair follicles and nervous system 124

and

125

Cerebral

organoid

iPSCs Coculture in plate Research of human neuronal cells migration 126

iPSCs Culture in spinning bioreactor Similar transcriptome and response to

inflammatory stimulation

127

iPSCs Culture in spinning bioreactor Repeatable realization of cell diversity 128

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Bone organoids are 3D self-organizing bone microtissues that can

reflect the complex bone physiological microenvironment and

structure as ideal models in vitro.

103

Bone microenvironment

recapitulation is the theoretical basis of bone organoid formation.

Functionally, the bone microenvironment mainly consists of three

units, i.e., bone formation, bone resorption and hematopoiesis.

There are several pivotal steps in establishing bone organoids

(Fig. 5b). Firstly, stem cells are isolated for diﬀerentiation. Then,

for the growth and diﬀerentiation of cells, biomaterials are

introduced as a matrix. Finally, some construction methods are

used to construct various organoid models, including woven bone

organoids, bone marrow organoids, cartilage organoids and

trabecular bone organoids.

104

Because of their high biomimetic eﬃcacy from the cellular

level to organ level, organoids have shown great potential in

human disease modelling. Organoids cultured from patient-

derived cells can achieve personalized treatment and perform

drug development, evaluation and screening.

105

Moreover,

organoids with complex 3D structures and specific functions

can be used in tissue engineering and regenerative medicine.

3.2.3 Assembloids. Assembloids were defined by Pas-ca

et al. as self-organizing cellular systems resulting from the

combination of a type of organoid with another type of orga-

noid or with diﬀerent specialized cell types, resulting in their

integration.

129

However, some researchers may define assem-

bloids as some type of organoid. As an example, Vogt claimed

that assembloids are organoids generated by spatially organiz-

ing multiple cell types.

130

It should be noted that we adopt the

definition of assembloids as a combination of diﬀerent types of

organoids in this work. Assembloids are more advanced

organoid-based cellular aggregates produced by a variety of cell

types with a spatial organization structure, which can integrate

diﬀerent types of organoids.

131

Compared with organoids,

assembloids can better achieve the complexity of the micro-

environment in vitro and the specificity of tissue structure,

as well as maintain the stability of cell genetic information.

Fig. 5 (a) Method for the construction of liver organoids and tumors from healthy livers. Isolated cells or tissues are seeded in suitable matrix scaﬀolds.

After 1–2 weeks of amplification, liver organoids are formed, and liver tumors are formed after 3–4 weeks. Reproduced with permission.

102

2021, Elsevier B.V. (b) Schematic diagram of the process for the biofabrication of bone organoids.

104

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They provide a new solution to overcome the shortcomings of

organoids, such as their single-cell type, lack of vascular system

and complex interstitial structure.

The successful construction of assembloidsismainlyachieved

by suspension culture, matrix culture, 3D bioprinting or organ-on-

a-chip technology, which are similar to the methods of organoid

construction. Suspension culture is the earliest method, which is

simple and time-consuming. It cannot provide matrix compo-

nents and a vascular system for cells, leading to an unstable

assembly structure and poor permeability in the growth process.

Alternatively, the matrix culture method can provide a matrix that

is rich in growth factors and provides mechanical support and

necessary substances for growth for the self-organization process

of cells. In addition, it can make the assembloid structure more

complete and plays a role in prolonging the cell culture cycle. 3D

bioprinting technology and organ-on-a-chip technology have

shown great potential in a wide area of biofabrication, including

the construction of assembloids, in which chemical and physical

signals can be applied and controlled more accurately. These

emerging technologies enable assembloids to achieve more

complex, complete, and delicate physiological structures,

132

better

compatibility

133

and higher throughput in vitro biomimetic cul-

ture. Further discussion is presented in Section 3.3.

In addition to the necessary physical and chemical stimula-

tions, the methods for the assembly of cells are also crucial for

the formation of assembloids. Diﬀerent protocells are used in

diﬀerent assembly methods. The common assembly methods

include spontaneous assembly and directed assembly. Sponta-

neous assembly is a process that mainly involves the sponta-

neous growth of stem cells to achieve assembly, while directed

assembly involves coculturing a variety of highly diﬀerentiated

cells in proportion to achieve assembly.

134

At present, diﬀerent assembloids have been constructed,

such as brain,

135

heart,

136

islet,

137,138

and tumor

139

assem-

bloids. For example, iPSCs were encapsulated in microcapsules

by microfluidic electrospray technology to eﬀectively form

brain region-specific organoids, which were then fused and

assembled into brain assembloids.

140

Brain assembloids can

grow and function well and are composed of cortical, hippo-

campal and thalamic organoids, indicating that they have

significant potential for a wide range of applications in the

neurological and biomedical fields. In addition, iPSCs are used

to generate 3D cortical and striatal organoids, which can be

assembled to form the corticostriatal circuitry of the forebrain

(Fig. 6a).

135

The neural circuit formed by the complete assembly

of the cortex, spinal cord and skeletal muscle can also be

cultured in vitro for up to 10 weeks, and control of muscle

contraction by the cortex can be simulated. The construction of

these models is of great significance for the study of neurode-

velopment and mental illness.

141

For the construction of heart

assembloids, PSCs are seeded on ultralow attachment plates

and cultured under chemical stimulation. Cardiac diﬀerentia-

tion and spontaneous assembly were induced to form heart

assembloids (Fig. 6b).

136

3.3 Integration platforms

Existing strategies for biomimetic cell culture have made great

progress in the simulation of spatial, chemical, and physical

microenvironments. However, the structural complexity, func-

tional diversity, and complicated communications at diﬀerent

levels (cell, tissue, and organ) are still insuﬃcient compared

with that in vivo. With the rapid development of 3D bioprinting

technology and microfluidic chip technology, they may serve as

platforms to improve existing biomimetic cell culture strategies

Fig. 6 (a) Schematic diagram of region-specific neural culture and assembly simulating human brain development in vitro. Reproduced with

permission.

135

permission.

136

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such as microcarriers, spheroids and organoids.

142

3D bioprint-

ing is manufacturing technology for the precise allocation of

biomaterials containing cells to construct complex living tis-

sues or artificial organs with specific functions.

143

It can be

used to print larger-sized complex constructs based on cellular

aggregates. Microfluidic chip systems are potent for culturing

living cells in micron-level chambers under continuous flow

conditions.

144

They can also be used to enable multiple biomi-

metic structures, such as organoids, to communicate with each

other to achieve more complicated biomimetic structures.

These integrative platforms can control the assembly of cellular

aggregates in a larger spatial range and improve the physiolo-

gical relevance of the models.

3.3.1 3D bioprinting technology. As additive manufactur-

ing technology, 3D bioprinting has great application prospects

in the construction of 3D spatial niches for cells in terms of

spatial geometry, compositions and spatial distribution of cells

and ECM. 3D bioprinting can give rise to biomimetic 3D

scaﬀolds with a uniform distribution of cells or cellular aggre-

gates.

145

There are diﬀerent bioprinting technologies based on

the basic working principle of manufacturing functional tissue

structures, including inkjet-based bioprinting,

146–148

laser-

assisted bioprinting,

149,150

extrusion-based bioprinting,

151,152

acoustic bioprinting,

153–155

photocuring-based bioprinting,

156–159

and magnetic bioprinting.

160–162

Table 3 presents a summary of

the main applications of these bioprinting technologies in

biomimetic cell culture with a brief description of each techno-

logy. These bioprinting strategies can achieve the requested

additive manufacturing goals and tissue manufacturing via

single or multiple uses, as shown in Fig. 7. As an example,

magnetic 3D bioprinting is used to form cellular aggregates in a

noncontact manner, where the cell medium is exposed to an

external magnetic field after mixing with a paramagnetic

buffer.

160

Nanoparticles can also be used to magnetize cells,

and thus they can be manipulated to suspend from the surface

into the culture medium to form cellular aggregates.

161

Magnetic

3D bioprinting has become an exocrine gland culture platform

with non-heterogeneity, high expansibility and reproduci-

bility.

162

In addition to bioprinting methods, bioink is another

essential part of the bioprinting strategy. Bioinks are blends

of cells and materials used for printing in 3D bioprinting

technology.

163,164

3D bioprinting has been extensively employed

to fabricate various porous scaﬀolds using bioinks from natural or

synthetic biological materials toward biomimetic cell culture and

tissue engineering.

As discussed above, microtissues, including cell-containing

microspheres and cellular aggregates such as spheroids, orga-

noids and assembloids, are superior to individual cells in terms

of biomimetic culture. Therefore, 3D bioprinting using bioinks

containing microtissues will have significant advantages over

regular 3D bioprinting using bioinks containing individual

cells. In the construction of the spatial microenvironment, it

not only realizes the construction of spatial geometry but also

precisely controls the compositions and spatial distribution of

cells and the matrix. For example, DLP bioprinting technology

was used to print microtissues from chondrocytes and cartilage

acellular matrix to form an auricular shape (Fig. 8a). 3D

bioprinting on a needle array, known as Kenzan bioprinting,

was used to fix spheroids on an array of microneedles and

manufacture tabular and tubular tissue constructs, as shown in

Fig. 8b.

198,199

Heo et al. printed spheroids formed by cocultured

MSCs and HUVECs by aspiration-assisted bioprinting

technology.

196

The printed tissue constructs have high spatial

structure stability and cell activity. Organoids or assembloids

have also been applied in 3D bioprinting (Fig. 8c) to control the

spatial position of cellular aggregates on a larger scale and

construct more complicated tissue structures. This process is

called bioprinting-assisted tissue emergence (BATE).

132

In the

BATE process, organoids or individual cells can be used as 3D

biological printing units, which can be forced to fuse and

recombine according to the preset shapes and sizes by 3D

printing. By utilizing this universal strategy, large-scale

complex cell constructs (such as the gut, Fig. 8d) can be printed

with particular cell types to generate tissue–tissue interactions

by simulating the process of native organ development or

homeostasis.

Additionally, the combination of spheroids and 3D bioprint-

ing technology improves the accuracy of cell regulation, which

simulates the cellular microenvironment more precisely, espe-

cially the chemical microenvironment.

200

To construct human

hypertrophic scars in vitro, Bin et al. developed a new bioink

containing cellular aggregates of fibroblasts, and then prepared

a scar tissue model with a precise arrangement and multiple

functions using bioprinting technology.

201

This model can not

only be used to study the key mechanisms of hypertrophic

scarring but also to rapidly search for drug targets and study

new optimal treatments. In addition, using 3D bioprinting

technology, liver organoids undergo more advanced assembly

and fusion, hepatocyte diﬀerentiation, albumin synthesis, liver-

specific enzyme activity, and a highly biomimetic polarization

phenotype.

188

These results prove that the bioprinting of 3D

cellular aggregates can achieve more complex regulation of

biochemical factors than the bioprinting of cells.

In terms of the physical microenvironment, studies have

shown that bioinks containing compacted cellular aggregates

exhibit higher yield stress than slurries of individual cells,

enabling the printing of more mechanically stable

structures.

202

Daly et al. developed a bioprinting method in

which spheroids could move within a shear-thinning hydrogel,

which could self-heal to localize the spheroids.

203

High cell-

density microtissues with specific shapes could be formed

by directed fusion between spheroids within the hydrogel.

By bioprinting this hydrogel containing spheroids composed

of iPSC-derived cardiomyocytes and cardiac fibroblasts, it was

found that the culture system supported cell diﬀerentiation,

proliferation, and migration, allowing the growth of multi-

cellular structures in prescribed 3D patterns, successfully

mimicking the global reduction in contractility that occurs

after myocardial infarction.

3.3.2 Microfluidic chip technology. Microfluidics is a tech-

nique for precisely handling small amounts of fluid in the form

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Table 3 Application of 3D bioprinting in biomimetic cell culture

Method Principle Cell type Material Printing structure Results Ref.

Inkjet-based

bioprinting

Division of bioink into

droplets and print layer by

layer

Fibroblasts Alginate, silk fibroin Tubular constructs Long-term cell culture in vitro 166

Fibroblasts Alginate, surfactants Hollow cell-containing

channels

Long-term cultures of larger samples 167

Fibroblasts Sodium alginate Cell-laden microsphere Construction of circulatory system and

reduction of cell sedimentation and

aggregation

168

Corneal epithelial/endothelial

cells

Poly-e-lysine, gellan gum Porous bitmaps Favor of the growth of monolayer cells 169

Type I and II alveolar cells, lung

fibroblasts, lung endothelial cells

Collagen Progressive deposition

of each layer of alveolar

tissue

Construction of the alveolar barrier

models

170

Laser-

assisted

bioprinting

Vaporization of the metal

film depositing on bioink

by laser; ejection of bioink

droplets

Stromal cells from apical papilla/

HUVECs

Collagen I, tricalcium silicate Disc Osteogenic diﬀerentiation and

angiogenesis

171

HUVECs N.A. Deposition on

biopaper

Production of vascularized bone

structure

172

MSCs Rat collagen I, nano HA Disc, ring Bone repair 173

ESCs, ADSCs Human laminin, collagen I Layer-by-layer printed

stacking structures

Simulation of corneal tissue structures 174

Extrusion-

based

bioprinting

Extrusion of bioink in the

syringe and cross-link

Fibroblasts GelMA, silicate nanoparticles Hollow cylinders Maximization of printability and

shape fidelity

175

HUVECs/breast cancer cells/

fibroblasts

GelMA, alginate core, sheath

microfibers

Hollow constructs with

curved and straight

channels

Favor of cellular proliferation and

spread

176

Muscle progenitor cells Acellular gelatin hydrogel, PCL Multilayer structure

containing

microchannels

Excellent cell proliferation and

diﬀerentiation

177

Dermal papilla cells/

keratinocytes

GelMA/gelatin Filament segments

containing hollow

channels

Construction of the intestinal villi/

human hair follicles structure

178

Small intestinal organoids Collagen I Tube constructs Control of tissue self-organization on

a larger scale

132

Acoustic

bioprinting

Movement of cells in dif-

ferent directions by sound

waves

Fibroblasts GelMA Pyramid structure Construction of tumor microtissue

invasion model

179

MSCs Pluronic F-127, Matrigel Cylinders consisting of

96 droplets per layer

Control of droplet size by changing

acoustic frequency

180

Colorectal organoids GelMA Single, line, cross, and

triangle patterns

Reestablishment of tumor structures

and simulation of tumor invasion

181

Photo-curing

based

bioprinting

Photocuring of bioinks by

light projection

Fibroblasts GelMA Lattice structures Production of steak-type cultured

meat

182

HUVECs CS, acrylamide Nose, ear, lattice

structures

Provision of bioactive sites for cell

growth

183

HUVECs GelMA Lattice structures Excellent biocompatibility for cultur-

ing cells in vitro

184

MSCs GelMA Cylinders Cartilage regeneration 185

MSCs GelMA, LAP, New Coccine Grid structures Excellent shape fidelity, eﬃcient and

low-cost cell culture strategy

186

Microtissue of chondrocytes on

acellular matrix microparticles

GelMA 3D auricular model Accurate assembly of organ building

blocks and increase in cell viability

187

Liver organoids GelMA Cylindrical organoid-

laden constructs

Reproduction of liver-specific ammo-

nia detoxification function

188

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of discrete droplets.

205–208

It can meet conditions such as a high

surface volume ratio and shear stress, and also realize the

control of physical or chemical conditions for the growth

microenvironment.

The cellular microenvironment, including cytokines, ECM,

physical cues and cocultured cell types, can be integrated in the

microfluidic platform (Fig. 9a), which supports cell growth,

metabolism, proliferation, diﬀerentiation, apoptosis and other

physiological activities. Recently, microfluidic technology has

made great progress in the construction of a biomimetic tumor

microenvironment (TME).

209

Chung et al. constructed a bio-

mimetic TME using a microfluidic platform to achieve the

coculture of tumor cell lines with primary fibroblasts (Fig. 9b).

210

The biomimetic culture system could be used to directly observe

angiogenesis induced by tumor–matrix interactions and to

reconstruct simultaneous angiogenesis and lymphangiogenesis

induced by tumor–matrix interactions and TME-simulating

extrinsic factors. The microenvironment of steatohepatitis

could also be constructed on the microfluidic chip (Fig. 9c). It

realized the steatohepatitis-related interactions of multiple

cells, multiple biochemical factors and matrices.

211

To precisely

control the oxygen tension in the cellular microenvironment,

Gao et al. fabricated a microfluidic device using oxygen-

permeable and water-impermeable materials.

212

By incorporat-

ing a gas-permeable zone in the top of the cell culture chamber

and equipping it with a tank of chemical caramel alcohol for

oxygen consumption, local control of the oxygen tension in the

cell culture was achieved. Integrated microfluidic devices could

be used to produce alginate microgels that encapsulate

cells and to gently separate the microgels from immiscible oil

solutions, resulting in a significant increase in cell survival.

213

Given the importance of chemical signals to cells, it is essential

to efficiently deliver related signal molecules such as biological

signal peptides in the microfluidic platform. Zhao et al. con-

structed GelMA microspheres integrated with biological signal

peptides in a microfluidic system, realizing the effective deliv-

ery of integrated biological signal peptides.

214

The system

constructed an accurate chemical microenvironment, which

substantially promoted the osteogenic differentiation of MSCs

and the vascularization of HUVECs in vitro.

Microfluidics have been used to regulate the biomimetic cell

culture environment in real time, such as controlling the

delivery of oxygen and nutrients, increasing the concentration

of metabolites and secretory factors, and regulating the

mechanical stimulation applied to cells.

216

For example, phy-

sical cues have been applied to finely tune cell culture channels

to reconstruct the spatial and physical microenvironment.

217

Microfluidic chip technology has also provided a potent plat-

form to study the communications between diﬀerent types of

cells by restricting the cells to diﬀerent chambers, between

which the exchange of signal molecules is allowed.

215

Microfluidic chip technology has been employed to estab-

lish in vitro microscopic physiological models, which are

known as organs-on-a-chip. Various in vitro physiological

models in microfluidic platforms have been developed to study

pathogenesis and pharmacology, aiming at overcoming severe

Table 3 (continued )

Method Principle Cell type Material Printing structure Results Ref.

Magnetic

bioprinting

Guidance of the magne-

tized cells into 3D patterns

by mild magnetic

Fibroblasts Alginate, methylcellulose, polyacrylic

acid-stabilized magnetite nano-

particles of Fe

Cubes Remote control and navigate hydrogel

actuators

189

Dental pulp stem cells Gold and iron oxide nanoparticles

crosslinked by poly-L-lysine

3D spheroids Formation of biofunctional salivary

gland organoids

190

Kenzan

bioprinting

Using the microneedles as

temporary support to pro-

vide spatial organization

for the spheroids to merge

Spheroid composed of MSCs N.A. Tubular constructs Dispensation of spheroids according

to the size and shape of defect

191

Spheroid composed of

hepatocytes

N.A. Spheres Construction of a novel liver tissue-

like model

192

Spheroid composed of dermal

fibroblasts

N.A. Ring-like tissues Reestablishment of tendon-like struc-

tures under tensile culture

193

Spheroid composed of chon-

drocytes, endothelial cells and

MSCs

N.A. Tubular constructs Generation of tubular artificial

tracheas

194

Spheroid composed of fibro-

blasts, HUVECs, MSCs and

smooth muscle cells

N.A. Tubular structures Generation of artificial esophagus 195

Aspiration-

assisted

Bioprinting

Pick of cells to the speci-

fied position by aspiration

forces

Spheroid composed of MSCs,

HUVECs

N.A. Geometrical shapes in

a single layer

Osteogenic spheroids as building

blocks

196

Spheroid composed of ADSCs Sacrificial alginate hydrogel Single layer structure

of diﬀerent component

Simulation of the bone and cartilage

structures in natural tissues

197

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health risks such as cancer and COVID-19. For example, Liu

et al. proposed a microfluidic method for the large-scale

production of multi-size 3D tumors from human hepatocarci-

noma HepG2 cells and human glioma U251 cells with a

biomimetic TME, which were used to monitor the tumor

response in diﬀerent fluid microenvironments and test multi-

ple anticancer drugs.

218

It provides an eﬃcient and low-cost

method for the development of anticancer drugs. To investigate

the underlying pathogenesis of COVID-19, Zhang et al. cultured

human alveolar epithelial cells, pulmonary microvascular

endothelial cells, and immune cells in a microfluidic chip to

mimic alveolar capillary barrier damage and the pulmonary

inflammatory response after SARS-CoV-2 infection.

219

The

microfluidic chip device consisted of an upper alveolar epithelial

channel and a lower pulmonary microvascular endothelial chan-

nel, and the cells were cocultured under perfused media flow,

thereby simulating the spatial structure of the cellular microenvir-

onment and the chemical microenvironment by cytokine secretion

(Fig. 10a). Guo et al. also studied the intestinal response to viral

infection in a microfluidic chip.

220

The upper channel cocultured

intestinal epithelial Caco-2 cells and HT-29 cells to establish the

intestinal barrier. The lower channel was cocultured with HUVECs

and immune cells to simulate the microvascular endothelium

(Fig. 10b). These organs-on-chips provided potent tools for testing

drugs and exploring disease mechanisms.

In addition to individual cells, microfluidic chip technology

can also be used to integrate cellular aggregates, especially

organoids,

221

to greatly improve the complexity and accuracy of

organ models in vitro. For example, Liu et al. constructed a

multiorgan-on-a-chip system consisting of upstream ‘‘lung

organs’’ and downstream ‘‘brain organs’’ (Fig. 10c).

222

The

multiorgan microfluidic chip was fabricated using two PDMS

layers and a microporous membrane. The upstream ‘‘lung

organs’’ are organoids cocultured with a sandwich structure,

including tumor cells, pulmonary vascular endothelial cells,

immune cells, fibroblasts and bronchial epithelial cells. The

two sides of the vacuum channel simulated the respiratory

rhythm to reconstruct the occurrence of lung cancer. Alterna-

tively, the downstream ‘‘brain organs’’ are organoids composed

of two cerebral parenchymal chambers surrounded by vascular

channels with a bionic blood–brain barrier structure. Vascular

channels and parenchymal chambers communicate through

microporous membranes to achieve intercellular interactions

and allow tumor cells to penetrate the blood–brain barrier.

Using this multiorgan-on-a-chip system, the pathological pro-

cess of primary lung cancer to brain metastasis can be simu-

lated, which provides a new idea for the development of specific

drugs and conducive to more accurate and effective treatment.

In another example, liver, heart, and lung organoids were

combined in a modular manner and perfused with culture

Fig. 7 Schematic of bioprinting technology. (a) Inkjet bioprinting system based on thermal or voltage. (b) Laser-assisted bioprinting system.

(c) Extrusion-based bioprinting system, including pneumatic pressure, piston and screw auxiliary structure. (d) Acoustic bioprinting system.

(e) Photocuring-based bioprinting system, including stereolithography appearance (SLA) and DLP. (f) Magnetic bioprinting system. Reproduced with

permission.

165

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medium in a microfluidic chip system (Fig. 10d).

223

This model

revealed the cardiotoxicity of bleomycin, which was previously

believed to be non-cardiotoxic by simple models such as heart

cells and organoids. The cardiotoxicity of bleomycin is gener-

ated as a result of cytokine-mediated interactions between the

lung and heart tissues. This is a typical example that indicates

the great significance of microfluidic chip technology as an

integrated platform that is superior to individual organoids.

By combining organoids with microfluidic technology, a more

complex and accurate cellular microenvironment can be

created, and more precise in vitro physiological models can

be established. In addition to the liver–heart–lung organoids-

on-a-chip described above, several other multiorgan-on-a-

chip systems have also been developed, such as liver–heart

organoids-on-a-chip

224

and gastrointestinal-liver organoids-on-

a-chip.

225

The liver-heart organoid on-a-chip system can be

used to evaluate the safety of antidepressants to heart tissue

after liver metabolism, which provides a new possibility for

predicting the side effects of antidepressant drugs.

224

Gastro-

intestinal-liver organoids-on-a-chip can be used to predict

how the two organs jointly respond to drugs.

225

These

multiorgan-on-a-chip systems include complex interactions

between different organs, providing a more realistic micro-

environment for cells than a single organ model. More infor-

mation about multiorgan-on-a-chip is available in a review by

Picollet-D’hahan et al.

226

Theoretically, various cellular aggregates, such as spheroids,

organoids and assembloids, can be incorporated into micro-

fluidic chip systems. The reasonable selection of cellular aggre-

gates and the design of microfluidic chips can provide a potent

strategy for biomimetic cell culture, based on which in vitro

tissue and organ models with biomimetic structures and func-

tions can be developed.

4. Challenges and future directions

With the growing understanding of the cellular microenviron-

ment and the emergence of new technologies, biomimetic cell

culture has reached an unprecedented height. Due to their

simple method and low requirements, the success rate of

conventional scaﬀold strategies is the highest among the

Fig. 8 (a) Schematic of DLP bioprinting of the microtissue bioink. The GelMA-microtissue bioink was printed in an auricular shape. Reproduced with

permission.

187

permission.

195

204

environment that allows multicell self-organization. Reproduced with permission.

132

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strategies. In contrast, the scaﬀold-free strategies of cellular

aggregates (spheroids, organoids and assembloids) are much

more complex and have higher requirements. In these strategies,

especially organoids and assembloids, the self-assembly of cells

involves complex cell communications and regulations, resulting

in lower success rates than that of conventional scaﬀold strategies.

Fig. 9 Construction of the cell microenvironment in a microfluidic chip. (a) Organ-on-a-chip relies on cells, biomaterials and culture systems to

reconstruct the microenvironment to replicate the key functional properties of tissues or organs. Reproduced with permission.

215

Elsevier B.V. (b) Establishment of the TME in vitro biomimetic model in a microfluidic platform. Reproduced with permission.

210

VCH. (c) Construction of the steatohepatitis microenvironment in a microfluidic chip. Abbreviations: Ch., channel. Reproduced with permission.

211

Fig. 10 (a) Construction of a SARS-CoV-2 infection model in vitro. Reproduced with permission.

219

configuration of SARS-CoV-2 infection on a chip device. Reproduced with permission.

220

pathological process of brain metastasis from lung cancer simulated by a multiorgan microfluidic chip. Reproduced with permission.

222

Elsevier B.V. (d) (i) Construction of an organ-on-a-chip system containing three types of organoids. (ii) Cell activity of cardiac organoids cocultured with

two other types of organoids on day 9. (iii) Cardiac organoid beating plots on day 9. Reproduced with permission.

223

Limited.

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For instance, Sachs et al. constructed breast cancer organoids with

asuccessrateofca. 80%.

227

Yan et al. constructed tissue organoids

from gastric cancer patients. The success rate of non-tumor tissue

organoids was ca. 90%, while that of tumor organoids was only

ca. 50%.

228

Tsao et al. constructed a non-small cell lung cancer

organoid with a short-term (o3months)successrateof74%.

229

3D bioprinting and microfluidic chip technologies as integration

platforms for biomimetic cell culture are still being developed. 3D

bioprinting using bioinks containing individual cells has been

widely applied in tissue engineering and possesses a high success

rate, while 3D bioprinting using bioinks containing cellular aggre-

gates (spheroids, organoids and assembloids) is more challenging

and has a lower success rate, as well as higher cost in terms of

time, money and labor.

230

Although individual cells and cellular

aggregates can be integrated into microfluidic chips to achieve

more advanced biomimetic cell cultures, this is still not suﬃcient

for engineering the meticulous and cumbersome conditions in

microfluidic chips. Moreover, due to the lack of uniform interna-

tional standards, there may be variations between individuals,

making their success rate lower. Their success rate is also limited

by the understanding of cell signaling pathways, the sensitivity of

sensing, analysis platforms, etc.

Conventional scaﬀold-based strategies, including solid scaf-

folds and hydrogels, can provide a 3D environment for cell

attachment and growth. Simple chemical and physical signals

can also be included in the structures. However, it is diﬃcult to

achieve a high cell density and precisely control the cell

distribution. It is also challenging to introduce gradients of

biochemical factors and complicated physical cues. In addi-

tion, nutrient supplementation is diﬃcult, especially for inner

cells when the constructs are large. New attempts and progress

in this field are being made to resolve these problems. Holland

et al. used a rotational internal flow layer engineering techni-

que to achieve a high cell density (10

cells mL

1

) and control

the distribution of cells in diﬀerent gel layers.

231

Wei et al.

constructed a bilayer hydrogel, which can achieve a biomimetic

gradient of composition, structural pore size, and biochemical

cues (kartogenin).

232

A smaller pore size (150–200 mm) and

higher concentration of kartogenin can induce the formation of

more chondrocytes, while a larger pore size (200–300 mm),

lower concentration of kartogenin and the presence of b-TCP

can induce more osteogenic differentiation.

Scaﬀold-free strategies, including spheroids, organoids and

assembloids, represent a significant step forward in the frontier

of biomimetic cell culture. They can achieve a biomimetic

spatial environment for cells with a high cell density and

suﬃcient cell–cell interactions. Moreover, gradients of bio-

chemical factors, oxygen and nutrients can also be formed in

cellular aggregates. Hubert et al. found that tumor organoids

generated nonlinear gradients of stem cell density and

hypoxia.

233

However, it is difficult to accurately control the

surrounding microenvironment of cellular aggregates, and it

cannot well reproduce the complex and dynamic microenviron-

ment in the process of organ or tissue development. Mean-

while, mature vascularization has not been achieved with these

cellular aggregates.

234

Thus, nutrient supplementation is still a

problem with an increase in the size of the aggregates. It was

found that 4 mm brain organoids began to undergo apoptosis

after approximately 100 days.

235

In addition, cellular aggregates

have variability and heterogeneity in terms of morphology and

function for different culture batches.

236

Although assembloids

increase the complexity and diversity in cell–cell and cell–

matrix interactions compared with individual organoids, infor-

mation exchange and functionality remain inadequate.

141

Recently, the converging strategies of cellular aggregates and

3D bioprinting or microfluidic chip technologies have shown

great potential in this field.

237,238

For example, Gong et al.

developed a controllable perfusion microfluidic chip to pre-

cisely control the fluidic shear stress and oxygen concentration

distribution in the process of culturing retinal organoids.

237

3D bioprinting and microfluidic chip technologies can serve

as integration platforms for diﬀerent forms of biomimetic cell

culture, especially cellular aggregates, which allows the con-

struction of complex heterogeneous multi-tissue/organ struc-

tures. Also, interactions among multiple tissues and organs can

be established. Moreover, using these integration platforms,

high-throughput preparation and measurement are possible

through precise regulation of chemical and physical signals.

239

In general, multiorgan-based bioprinting and microfluidic

chips are currently the most promising systems of biomimetic

cell culture. Microfluidic chips have significant advantages

in controlling specific organizational structures and chemical

gradients and can achieve a high-level biomimetic micro-

environment.

240

However, integration platforms are also limi-

ted by some of the challenges faced by cellular aggregates, such

as nutrient supplementation and reproductivity. Recently, an

oxygen detection system that can detect the oxygen pressure in

real time was reported.

241

It could deliver oxygen when the

oxygen concentration decreased, which may facilitate oxygen

supply and diﬀusion in cellular aggregate culture. Nevertheless,

the resolution of nutrient supplementation ultimately relies on

the introduction of functional blood vessels.

242

In addition, to

overcome the issue of heterogeneity and variability, it is essential

to standardize the production of cellular aggregates and introduce

precise biosensor elements and automation systems.

243,244

Although biomimetic cell culture has made great progress,

there is still a long way to go to precisely engineer the micro-

environment of cells. In the future, the emergence and integra-

tion of new technologies may boost the pace of biomimetic cell

culture. For example, the application of gene editing technology

in cellular aggregates provides great potential for the develop-

ment of research, disease modelling and personalized medicine.

DuringtheCOVID-19pandemic,therapiduseofintegration

platforms to explore mechanisms and treatments has demon-

strated the advantages of using CRISPR-Cas9 technology

245

for

cellular aggregates. This technology has completely changed the

modelling of human organs and tumors, thereby deepening our

understanding of embryogenesis, organogenesis and tumori-

genesis.

246

Moreover, nanoscale biomaterials, such as nano-

particles, nanocapsules and nanofibers, are expected to be used

as functional additives to regulate physiological and metabolic

activities in biomimetic cell culture. Functional nanomaterials also

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help to improve the cell density of stem cell spheroids, as well as

the activity, renewal ability, proliferation ability, diﬀerentiation

ability and paracrine eﬀect of stem cells. The control of the spatial,

chemical and physical microenvironment in an in vitro model is

the key to realizing biomimetic cell culture. Therefore, exploring

the eﬀects of spatial structure, chemical signals and physical cues

on cells is a prerequisite for achieving controllability. The combi-

nation of biomimetic cell culture and automation technology is an

interdisciplinary task and may be a good choice to develop

reproducible in vitro models in the future.

5. Conclusion

How to maintain or recapitulate the native key features of cells,

such as morphology, behavior, metabolism, and function,

in vitro is crucial for a wide range of fundamental biomedical

research and applications, especially in disease modelling and

pathological studies, drug development and evaluation, tissue

engineering and regenerative medicine. The basic principle of

biomimetic cell culture is to rebuild a microenvironment for

cells in vitro comparable to their native microenvironment

in vivo. The main task lies in the reoccurrence of the compli-

cated microenvironment in vitro by using appropriate materials,

signals and manufacturing technologies. The complicated micro-

environment of cells can be roughly simplified into three aspects,

i.e.,spatial,chemicalandphysicalfeatures.Spatialstructuressuch

as dimension and pore size can aﬀect cell adhesion, migration,

and proliferation. The pore size in the spatial microenvironment

can aﬀect the direction of cell diﬀerentiation, and the pore

orientation may directly aﬀect the direction of cell migration.

The composition and spatial distribution of cells and the ECM

are also significant in the spatial microenvironment. Various

biochemical factors, such as interleukin, TGF-b,HGF,BMP-4

and O

, play a major role in the chemical microenvironment. They

control the growth and metabolism of cells and have profound

eﬀects on the fate of cells by regulating gene transcription and

protein synthesis and secretion. Physical cues in the microenvir-

onment are also crucial factors that influence cell fate. Internal

stress, such as fluid shear stress, hydrostatic pressure and oxygen

partial pressure, and external stimuli may aﬀect cell behaviors by

activating specific signaling pathways. The spatial, chemical, and

physical signals in the cellular microenvironments play a synergis-

tic role in biomimetic cell culture in vitro.

This has inspired a variety of strategies for biomimetic cell

culture involving a 3D spatial environment, specific biochem-

ical signal molecules, and appropriate physical cues. Eﬀorts

have been made to achieve biomimetic cell culture in recent

decades with fast-growing development from conventional

scaﬀold-based methods to self-organized multicellular aggre-

gates such as spheroids, organoids and assembloids. Recently,

the development of 3D bioprinting and microfluidic chip

technologies has provided potent platforms to achieve higher-

level biomimetic cell culture. Conventional scaﬀold-based

strategies are easy to conduct, but they only involve simple

physical or chemical cues and limited cell density and

distribution. In comparison, cellular aggregates have a higher

cell density and higher-level biomimetic microenvironment,

although they suﬀer from issues of nutrient supplementation

and reproductivity. 3D bioprinting and microfluidic chip tech-

nologies integrated with cellular aggregates have reached a new

height in biomimetic cell culture. Despite the current issues of

variability and heterogeneity of cellular aggregates, they are

promising strategies for biomimetic cell culture by establishing

standards and introducing intelligent and automated systems.

In the era when animal experiments are no longer the standard,

biomimetic cell culture in the future will play a great role in

disease modelling, drug development, tissue engineering and

regenerative medicine with the advantages of high throughput,

low cost, high precision and low ethical risk.

Author contributions

Qiuchen Luo, Zhaoying Wu: data curation, visualization, meth-

odology, writing – original draft, writing – review & editing.

Keyuan Shang, Jing Zhu, Abeer Ahmed Qaed Ahmed, Chixiang

Huang: data curation, visualization, writing – review & editing.

Tiefeng Cao: resources, writing – review & editing, funding

acquisition. Lin Xiao: conceptualization, methodology, writing –

review & editing, supervision, project administration, funding

acquisition.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

The authors acknowledge the National Natural Science Founda-

tion of China (52173151, 82002734, 51803067), the Natural

Science Foundation of Guangdong Province of China (2021A15-

15011084, 2019A1515110312), the Fundamental Research

Funds for the Central Universities (22qntd1302) and Shenzhen

Outbound Postdoctoral Scientific Research Funding (SZBH202108)

for their financial support. The authors thank Prof. Antonella

Forlino from the Department of Molecular Medicine, Biochemistry

Unit, University of Pavia, 27100 Pavia, Italy, for her valuable

contribution in editing the current review.

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Stem cell-based therapy is a novel technique used to reconstruct the ocular surface of patients with limbal stem cell deficiency (LSCD). However, the reported stem cell therapies for LSCD are limited to two-dimensional (2D) culture systems, which cannot mimic the native microenvironment in which stem cells reside in vivo. Herein, an injectable photocrosslinkable hydrogel was developed based on porous gelatin methacryloyl (GelMA)/silk fibroin glycidyl methacrylate (SilMA), which encapsulated limbal stem cells (LSCs) to reconstruct ocular surfaces. The porous GelMA/SilMA hydrogels demonstrated excellent transparency and good adhesion. More importantly, this hydrogel maintained viability, supported the adhesion of encapsulated rabbit LSCs and promoted their migration in vitro in three-dimensional (3D) culture environments. Proteomic analysis confirmed that the porous GelMA/SilMA hydrogel 3D cell culture system maintained both the self-renewal and differentiation of the LSCs. Furthermore, histological analyses demonstrated that the porous GelMA/SilMA hydrogel encapsulating LSCs effectively promoted the re-epithelization of LSCD. Porous hydrogels were subjected to simplified serial cell culture operations to construct an ocular surface with a uniform cell distribution. These findings provide valuable insights into stem cell therapies and regenerative medicine for ocular surface reconstruction.

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The bioassembly of layered tissue that closely mimics human histology presents challenges for tissue engineering. Existing bioprinting technologies lack the resolution and cell densities necessary to form the microscale cell-width layers commonly observed in stratified tissue, particularly when using low-viscosity hydrogels, such as collagen. Here we present rotational internal flow layer engineering (RIFLE), a novel, low-cost biofabrication technology for assembling tuneable, multi-layered tissue-like structures. Using high-speed rotating tubular moulds, small volumes of cell-laden liquids added to the inner surface were transitioned into thin layers and gelled, progressively building macroscale tubes composed of discrete microscale strata with thicknesses a function of rotational speed. Cell encapsulation enabled the patterning of high-density layers (108cells/ml) into heterogenous constructs. RIFLE versatility was demonstrated through tunica media assembly, encapsulating human smooth muscle cells in cell-width (12.5µm) collagen layers. Such deposition of discrete microscale layers, facilitates the biofabrication of composite structures mimicking the nature of native stratified tissue. This enabling technology has the potential to allow researchers to economically create a range of representative layered tissue.&#xD.

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Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state‐of‐the‐art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large‐scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life‐like human tissue models that provide a platform to answer health‐based scientific questions.

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Bone is one of the key components of the musculoskeletal system. Bone and joint disease are the fourth most widespread disease, in addition to cardiovascular disease, cancer, and diabetes, which seriously affect people’s quality of life. Bone organoids seem to be a great model by which to promote the research method, which further could improve the treatment of bone and joint disease in the future. Here, we introduce the various bone and joint diseases and their biology, and the conditions of organoid culture, comparing the in vitro models among 2D, 3D, and organoids. We summarize the differing potential methods for culturing bone-related organoids from pluripotent stem cells, adult stem cells, or progenitor cells, and discuss the current and promising bone disease organoids for drug screening and precision medicine. Lastly, we discuss the challenges and difficulties encountered in the application of bone organoids and look to the future in order to present potential methods via which bone organoids might advance organoid construction and application.

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Article

Jul 2023

Tissue-engineered articular cartilage constructs are currently not able to equal native tissues in terms of mechanical and biological properties. A major cause lies in the deficiency in engineering the biomechanical microenvironment (BMME) of articular chondrocytes. In this work, to engineer the BMME of articular chondrocytes, heterogeneous hydrogel structures of gelatin methacrylated (GelMA) containing differential-stiffness domains were first fabricated, and then periodic dynamic mechanical stimulations were applied to the hydrogel structures. The chondrocyte phenotype of ATDC5 cells was enhanced as the spatial differentiation in stiffness was increased in the hydrogel structures and was further strengthened by dynamic mechanical stimulation. It was speculated that the mechanical signals generated by the engineered BMME were sensed by the cells through the integrin β1-FAK signaling pathway. This study revealed the key role of the combined effects of differential and dynamic BMME on the chondrocyte phenotype, which could provide theoretical guidance for highly active tissue-engineered articular cartilage.

A controllable perfusion microfluidic chip for facilitating the development of retinal ganglion cells in human retinal organoids †

Article

Jul 2023
LAB CHIP

Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) have become a promising model in vitro to recapitulate human retinal development, which can be further employed to explore the mechanisms of retinal diseases. However, the current culture systems for ROs lack physiologically relevant microenvironments, such as controllable mechano-physiological cues and dynamic feedback between cells and the extracellular matrix (ECM), which limits the accurate control of RO development. Therefore, we designed a controllable perfusion microfluidic chip (CPMC) with the advantages of precisely controlling fluidic shear stress (FSS) and oxygen concentration distribution in a human embryonic stem cell (hESC)-derived RO culture system. We found that ROs cultured under this system allow for expanding the retinal progenitor cell (RPC) pool, orchestrating the retinal ganglion cell (RGC) specification, and axon growth without disturbing the spatial and temporal patterning events at the early stage of RO development. Furthermore, RNA sequencing data revealed that the activation of voltage-gated ion channels and the increased expression of ECM components synergistically improve the growth of ROs and facilitate the differentiation of RGCs. This study elaborates on the advantages of the designed CPMC to promote RO growth and provide a controllable and reliable platform for the efficient maturity of RGCs in the ROs, promising applications in modeling RGC-related disorders, drug screening, and cell transplantation.

Organs-on-a-chip: a union of tissue engineering and microfabrication

Article

Jan 2023

We review the emergence of the new field of organ-on-a-chip (OOAC) engineering, from the parent fields of tissue engineering and microfluidics. We place into perspective the tools and capabilities brought into the OOAC field by early tissue engineers and microfluidics experts. Liver-on-a-chip and heart-on-a-chip are used as two case studies of systems that heavily relied on tissue engineering techniques and that were amongst the first OOAC models to be implemented, motivated by the need to better assess toxicity to human tissues in preclinical drug development. We review current challenges in OOAC that often stem from the same challenges in the parent fields, such as stable vascularization and drug absorption.

Biomimetic cell culture for cell adhesive propagation for tissue engineering strategies

Abstract

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