[Part 1]

Research Background

Various types of stem cells have become the most attractive cell source in tissue engineering due to their ability for self-renewal and pluripotent differentiation. The regulation of stem cell activity is primarily achieved through the use of biomaterial scaffolds, facilitating the regeneration or repair of diseased and damaged tissues.

As stem cells grow within scaffolds, they perceive the scaffold as their microenvironment, responding by altering their behavior. Tissue engineering often employs traditional fixed-size scaffolds and injectable biomaterials. However, these methods face significant limitations in generating macroscopic functional tissues.

Therefore, precise control over scaffold characteristics and the development of methods to enhance cell-material interactions are crucial for the establishment of tissue engineering technologies based on clinical applications of stem cells.

Facing Challenges:

Hydrogels, owing to their excellent tunability and precise control over scaffold characteristics, play a crucial role in influencing cellular activities and are frequently employed in tissue engineering. They are a promising tool for studying cell-substance interactions or serving as cell carriers to promote tissue repair. However, their in vivo application in tissue engineering technologies still faces several challenges:

1.Traditional Hydrogels:

Prolonged cultivation often leads to increased cellular aging, alterations in the "stemness" of stem cells, or impacts on the secretion of paracrine factors.

2.Self-crosslinking Hydrogels:

Inducing self-crosslinking methods are based on specific hydrogel systems and may not be widely applicable to other hydrogel formulations.
During the gelation process, damage is inflicted on the encapsulated cells. Moreover, limitations in extracellular matrix and cell-cell interactions can affect the post-gelation cell viability.

Physical crosslinking makes hydrogels susceptible to mechanical compliance breakdown, while chemical crosslinking may be associated with a certain level of toxicity to the encapsulated cells.

New Approach:

3D Microcarriers:

● Possess excellent biocompatibility.

● Larger surface area facilitates interactions with drug molecules.

● Feature both external and internal pores, promoting effective transport of nutrients, oxygen, and waste.

● Provide a porous microenvironment for cells, shielding them from mechanical damage and better maintaining cell activity and functionality.

By loading mesenchymal stem cells onto 3D microcarriers to form microtissues and placing them in a 3D-printed framework, cultivation for several days induces the pre-assembly of tissue structures with specific functionalities. 

[Part 2]

Materials and Methods:

Utilize 3D printing technology to create a grid framework, prepare gelatin microcarriers, conduct cell culture, and prepare microcarriers loaded with MSCs. Load these microcarriers onto the grid framework. Evaluate cell activity, apoptosis, and cell functionality (trilineage differentiation, aging), perform secretome analysis, proteomic analysis, bioinformatics analysis, and conduct macroscopic and microscopic mechanical tests on the pre-assembled structure. Finally, carry out in vivo implantation experiments on rabbit joint cartilage defects in three groups and conduct ex vivo analyses.

Image 1. Schematic Diagram of the Overall Research Design 
 

[Conclusion]

3.1 Characterization and Functional Evaluation of Pre-Assembled Mesenchymal Stem Cells (MSCs) Compared to Non-Pre-Assembled Microcarriers and 2D Cells.

The MSCs grown in the pre-assembled structure maintain their stem cell phenotype, similar to non-pre-assembled microcarriers and 2D-cultured MSCs. Furthermore, they exhibit better maintenance of stem cell characteristics and suppression of cell aging. Secretome analysis of the conditioned media from cells cultured in the pre-assembled structure after starvation reveals activation of paracrine functions in MSCs, including recruiting factors, proliferation factors, immunomodulatory factors, and vascular growth factors.

Image 2. Characterization and Functional Evaluation of MSCs in the Pre-Assembled Construct Compared to Non-Pre-Assembled Microcarriers and Monolayer Culture 

3.2 Characterization and Functional Analysis of MSCs in 3D Constructs Assembled at Different Time Points

On Day 3, the formation of the pre-assembled structure is observed, and by Day 7, it exhibits the predefined shape. Removal of the framework on Days 7 and 14 does not alter the structure of the construct. Within the first 7 days of culture, cell viability remains relatively stable. However, by Day 14, cell proliferation is significantly inhibited, and cell death increases significantly. As the culture time increases, the extracellular matrix (ECM) gradually accumulates. By Day 7, the accumulation of ECM leads to the significant maturation of the 3D microtissue construct, providing satisfactory mechanical properties.

On the 7th day after construct assembly, the expression of MSC-related markers (SOX2, NANOG, and OCT4) is significantly upregulated and remains relatively stable on Day 14. Aging-related markers (P16, P21, and P53) are significantly downregulated starting from Day 3 of the pre-assembly process. MSCs cultured in the pre-assembled structure on Day 7 maintain optimal stemness and suppress the aging process, with paracrine functions increasing over time. 

Image 3. Characterization of MSCs in 3D Constructs Pre-Assembled at Different Time Points

3.3 Mechanism of the Successfully Pre-Assembled Functional Structure of 3D Microcarriers Loaded with MSCs

At 0, 3, 7, and 14 days, protein characteristic analysis was conducted on the pre-assembled construct, revealing 199 highly expressed proteins. Notably, on Day 14, there was a significant doubling of the quantity of highly expressed proteins, such as COL4A2 and SOD2. The highly expressed proteins are primarily ECM-related proteins, including collagen, fibronectin, and LOXL2. These proteins can serve as binding sites for matrix components or cell surface receptors, facilitating the formation of a complex 3D network through cell-matrix interactions.

Image 4. Mechanism of the Successfully Pre-Assembled Functional Construct of Microtissues

3.4 In Vivo Cartilage Repair Using the Pre-Assembled Construct

Macroscopic evaluation, histological assessment, and immunohistochemical analysis of cartilage defect repair revealed that the surface of the repaired cartilage defects using the 3D construct was smooth, nearly completely filled with regenerated tissue, and the newly formed tissue resembled the appearance of surrounding normal cartilage. The filled translucent cartilage exhibited histological characteristics similar to the surrounding normal cartilage. In contrast, the other two groups showed only partial tissue repair, with irregular surfaces, soft texture, and distinct defect boundaries. The repair tissue in these groups consisted of a mixture of fibrous and cartilage-like cartilage. The cartilage repair efficacy of the 3D construct group was superior to the other two groups.

Image 5. In Vivo Cartilage Repair Using the Pre-Assembled Construct 

In summary, in this study, the pre-assembly of 3D microcarriers containing mesenchymal stem cells (MSCs) using a grid framework and gelatin microcarriers achieved remarkable advantages in maintaining MSC phenotypic characteristics and stemness compared to non-pre-assembled microcarriers and 2D cell culture. The pre-assembled 3D functional tissue structure exhibited significant capabilities in inhibiting cell aging and enhancing paracrine functions. The mechanism of pre-assembly was found to be associated with extracellular matrix (ECM) accumulation, reinforcing cell-ECM interactions within the structure.

In a rabbit joint cartilage defect model, the pre-assembled 3D construct demonstrated strong chondrogenic induction, evident by the formation of translucent cartilage, which was not observed in the groups with individual microcarrier cell filling or the control group. Therefore, the pre-assembled construct holds promise as an alternative tissue engineering implant to facilitate in vivo repair of tissue defects.

This strategy of developing 3D macroscopic tissue structures allows for the optimization of stem cell functionality and the construction of biomimetic tissue-like organs, showing potential applications across various tissues and tissue engineering endeavors. 

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【CytoNiche】

Beijing CytoNiche Biotechnology Co., Ltd. was established by the research team of Professor Du Yanan from Tsinghua University School of Medicine, and was jointly established by Tsinghua University through equity participation. The core technologies were derived from the transformation of scientific and technological achievements of Tsinghua University. CytoNiche focuses on building an original 3D cell "smart manufacturing" platform, as well as providing overall solutions for the 3D microcarrier-based customized cell amplification process.

Products and services of CytoNiche can be widely used in the upstream process development of gene and cell therapy, extracellular vesicles, vaccines, and protein products. At the same time, it also has broad prospects for applications in the fields of regenerative medicine, organoids, and food technology (cell-cultured meat, etc.).

Our company has a R&D and transformation platform of 5,000 square meters, including a CDMO platform of more than 1,000 square meters, a GMP production platform of 4,000 square meters, and a new 1200 L microcarrier production line. The relevant technologies have obtained more than 100 patents and more than 30 articles about the technologies in international journals have been published. The core technology projects have obtained a number of national-level project support and applications.