Table 3 Applications of EVs derived from 3D cultured SCs

Bone marrow-derived MSCs

1

A diabetic mouse wound healing model

HBMMSC-EVs

3D-printed scaffold perfusion bioreactor

3D-EVs promoted the percentage of wound closure and the number of neovascularization, significantly improving wound healing, whereas there was no significant difference between the vehicle control and 2D-EVs

[143]

2

In vitro wound healing model; in vitro cellular aging model

HBMMSC-EVs

3D aggregation wave reactor

Compared to 2D-EVs, 3D-EVs promoted fibroblast growth and wound healing faster and significantly reduced the number of senescent SCs, exerting anti-senescence ability

[148]

3

In vitro model for angiogenesis and neurogenesis

HBMMSC-MVs

3D culture based on PEG hydrogel microwell arrays

3D-MVs significantly stimulated HUVEC tube formation than IBE-MVs and VEGFs, but their ability to stimulate neural stem cell differentiation and proliferation was less than that of IBE-MVs, although it was stronger than that of NGFs

[159]

4

In vitro trigeminal ganglia neurite growth, elongation and complexity assays

HBMMSC-EVs

3D bioreactor based on microcarriers

Compared with 2D-EVs, 3D-EVs promoted neurite growth more, induced neurite elongation, and significantly increased neurite branching and complexity

[160]

5

A well-established controlled cortical impact rat model of TBI

HBMMSC-Exos

3D collagen scaffold culture

There was no significant difference between 2D-Exos and 3D-Exos in improving the recovery of sensorimotor function, promoting angiogenesis in rats after TBI, but 3D-Exos enhanced spatial learning better than 2D-Exos

[161]

6

5XFAD mouse model

HBMMSC-EVs

3D aggregation culture using ultra-low attachment plate

Compared with the saline group, 3D-EVs treatment significantly improved cognitive ability, reduced amyloid plaque deposition and the amount of GFAP in the brain, slowing down the progression of AD in 5XFAD mice

[162]

7

SCI rat model

SD rats BMMSC-Exos

3D culture of GelMA-based hydrogels

3D-Exos was more effective than 2D-Exos in reducing inflammation and neuroglial scarring, and promoting nerve regeneration after SCI

[43]

8

In vitro wound healing model

BMMSC-EVs

A novel microcarrier-based vertical-wheel bioreactor

The bioreactor EV group closed scratches at a faster rate than the 2D-EV group

[139]

Umbilical cord-derived MSCs

9

Excision wound healing mouse model

HucMSC-EVs

3D spheroid culture using an orbital shaker

3D-EVs better promoted dermal fibroblast migration, improved reepithelialization of the epithelial layer, and facilitated wound closure in excision wound model mice than 2D-EVs

[147]

10

In vivo wound healing splinting rat model

HucMSC-Exos

3D spinner flask culture

Compared with 2D-Exos, 3D-Exos had a stronger effect on reducing the area of granulation tissue in vivo, promoting complete re-epithelization of the wound, and facilitating skin healing after injury

[163]

11

A rat knee osteochondral defect model

HucMSC-Exos

3D printed acellular cartilage extracellular matrix scaffold culture

3D-Exos had enhanced ability to modulate the microenvironment of the articular cavity compared to 2D-Exos, and when used in conjunction with microporous scaffolds it could better repair osteochondral defects

[164]

12

Cisplatin-induced AKI mouse model

HucMSC-Exos

The hollow fiber bioreactor

3D-Exos are more renoprotective than 2D-Exos in ameliorating cisplatin-induced AKI

[28]

13

APP/PS1 double transgenic mice with alzheimer's disease

HucMSC-Exos

3D graphene scaffold culture

3D-Exos reduced amyloid-β production and more dramatically improved the memory and cognitive deficits in AD mice, with a stronger therapeutic effect than 2D-Exos

[165]

14

A rat model of AMI

HucMSC-EVs

The hollow fiber bioreactor

3D-EVs were able to significantly inhibit cardiomyocyte apoptosis, promote angiogenesis, and improve cardiac function in rats with acute myocardial infarction with strong cardioprotective effects compared with 2D-EVs

[33]

15

Silica-induced silicosis mouse model

HucMSC-Exos

Microcarrier-based 3D dynamic culture

3D-Exos inhibited silica-induced pulmonary fibrosis and improved lung function

[166]

16

Tube formation assay; wound healing assay; TNF-α induced inflammation model; TGF-β induced fibrosis model

HucMSC-EVs

3D spheroid culture using StemFIT 3D® plate and polydimethylsiloxane (PDMS)-coated flask

The researchers verified the pro-angiogenic, pro-wound healing, anti-inflammatory, and anti-fibrotic effects of EV- derived in 2D culture, 3D culture and 3D culture stimulated with a combination of TNF-α and IFN-γ (TI), of which TI-intervened 3D culture enhanced the mentioned functions of EVs

[140]

17

In vivo rabbit cartilage defect model

UCMSC-Exos

The hollow fiber bioreactor

Defective cartilage treated with 3D-Exos showed more neo-tissue formation and better fusion of the surrounding hyaline cartilage, which was superior to 2D-Exos in cartilage repair

[142]

18

Migration and proliferation of murine fibroblasts in vitro

UCMSC-Exos

3D spheroid culture using the aggrewell system

Exosomes derived from 3D serum-free cultured UCMSCs significantly increased migration and proliferation of murine fibroblasts, with the potential to accelerate wound healing

[167]

19

CD14 + cell migration assay, coculture of 661W cells and MVs

Human UCBMSC-MVs

3D spheroid culture using the hanging drop protocol

3D-MVs inhibited the migration of CD14 + cells better and stimulated the secretion of signaling factors from 661W cells at a stronger rate than 2D-MVs

[168]

Other derived SCs

20

CCL4 in conjunction with employing alcohol- induced liver fibrosis model in mice

HESC-Exos

3D spheroid culture using ultralow attachment plate

Compared with 2D-Exos, 3D-Exos significantly accumulated in the liver of fibrotic mice, significantly reduced the expression of pro-fibrotic markers and liver injury markers, inhibited liver fibrosis, and restored liver function

[42]

21

The acute renal I/R injury mouse model

HPMSC-EVs

3D spheroid culture using ultralow attachment plate

Compared with 2D-EVs, 3D-EVs were able to counteract apoptosis and inflammatory responses more effectively, resulting in reduced tissue damage, improved renal function, and better protection against I/R progression

[41]

22

In vitro HUVEC migration assay and capillary-like formation assay

HAMSC-Exos

3D spheroid culture using ultralow attachment plate

Both 2D and 3D hAMSC-Exos at the same concentration induced capillary-like formation and endothelial cell migration, and there was no significant difference between the two

[169]

23

Ligature-induced periodontitis model, DSS-induced colitis model

HDPSC-Exos

3D spheroid culture using ultralow attachment dish

Compared to 2D-Exos, 3D-Exos exerted enhanced amelioration of periodontitis and colitis

[44]

24

In vitro dorsal root ganglia neuron sprouting assay

HDPSC-EVs

3D Fibra-Cell scaffold culture

3D-EVs generated by dynamic scaffold cultured cells showed a stronger ability to stimulate neuronal axon sprouting compared to 2D-EVs

[170]

25

6-OHDA -induced apoptosis in human dopaminergic neurons

HDPSC-Exos

3D microcarrier cell culture

3D-Exos significantly inhibited 6-OHDA-induced apoptosis in dopaminergic neuronal cells, showing neuroprotective properties, whereas no such effect was observed with 2D-Exos

[171]

26

SD rat alveolar bone defect model

HPDLSC-Exos

Collagen hydrogel-assisted 3D culture system

3D-Exos effectively induced alveolar bone regeneration and expression of osteogenic proteins Runx2 and OPN in SD rats

[145]