Recently, the team of Professor Jiang Qing from Nanjing University and the team of Professor Du Ya'nan from Tsinghua University jointly published a research paper entitled "A low dose cell therapy system for treating osteoarthritis: In vivo study and in vitro mechanistic investigations" in Bioactive Materials (IF: 14.593).

The study discovered that the use of mesenchymal stem cell (MSCs)-laden microcarriers prepared by 3D large-scale culture process (i.e., MSC microtissues) as a drug delivery platform for stem cell therapy could address a series of problems with traditional free stem cell administration in the treatment of osteoarthritis. It has the potential to reduce the cell dosage and injection frequency required for therapeutic applications, and the potential mechanism of action of MSC microtissues for osteoarthritis treatment was explored through secretome and transcriptome analysis.

[Study Background]

Osteoarthritis (OA) is a non-inflammatory degenerative disease characterized by pathological manifestations of articular cartilage degeneration and secondary bone hyperplasia. Its incidence in China is up to 15%, and in recent years, the onset of OA is gradually showing a "younger" trend. The clinical symptoms of OA include joint stiffness, swelling, pain, and periarticular muscle atrophy, which seriously affect the quality of life of patients.

Benefiting from the potential and easy availability of mesenchymal stem cells (MSCs) in terms of self-renewal, directional differentiation, and immune regulation, MSC-based stem cell therapy is widely used in the preclinical and clinical studies of osteoarthritis as an emerging regenerative medicine therapy, with initial success. In the article "Expert Consensus on Clinical Drug Treatment of Osteoarthritis" published in the "Chinese Journal of the Frontiers of Medical Science (Electronic Version)" in 2021, MSCs were listed as candidate drugs of biologics for the treatment of osteoarthritis.

However, the traditional delivery method using intra-articular injection of free MSC still suffers from a series of problems such as cell dosage, cell retention, cell survival, and integration into the host tissue, which limit the stability of its efficacy. Therefore, more effective cell formulations and delivery platforms should to be developed. Since 2014, the team of Professor Du Ya'nan from Tsinghua University School of Medicine pioneered the concept of "stem cell pharmacy" internationally. It uses 3D porous microcarrier composite stem cells for formulation and drug delivery, which has made a series of research progress in the fields of diabetes and its complications, osteoarthritis, liver fibrosis, etc.

[Study Content]

▲Figure｜Technical route design of this study

1. In Vitro 3D Culture to Form MSC Microtissues

The study results show that the application of CytoNiche 3D FloTrix® Microcarriers combined with a stirred bioreactor can enable the in vitro formation of MSC microtissues. Within a certain culture period, the stem cells in the microtissues maintained high viability (green fluorescence marks live cells, red fluorescence marks dead cells). The expression of stemness-related genes (Sox2, Oct4, Nanog) were up-regulated, and the senescence-related genes (P21, P16, P53) were downregulated. Compared with free MSCs injected independently, MSCs administered in the form of microtissues can be retained in mice for more than 21 days, showing higher viability.

2. MSC Microtissue for the Treatment of in Vivo OA Models

For the in vivo model presented in this article, an in vivo OA model is constructed by performing anterior cruciate ligament (ACL) transection in rats (Figure A). Combined with Micro-CT (μ-CT), radiological imaging, and histological analysis, the treatment effect of a single dose injection of MSC microtissue with 1 × 10⁵ initial cell count was comparable to that of a 4-dose injection of 1 × 10⁶ free MSC after comparison of different treatment methods (see the original paper for figures of imaging analysis and histological analysis).

[Application of MDCK Cells for Vaccines]

MDCK cells are considered to be one of the most suitable cell lines for the production of influenza A and B vaccines because of their high affinity to viruses, rapid proliferation rate, and resistance to mutations. MDCK originally requires adherent culture, but traditional two-dimensional planar culture is not conducive to gradual scale-up. With the discovery and use of microcarriers, this problem is gradually solved. Microcarriers can realize suspension culture of adherent cells, which can effectively increase the amount of cell culture and virus harvesting in one batch.

3. Investigation of the Mechanism of Interaction between MSC Microtissue and Chondrocytes

In this study, after obtaining cartilage tissue from donors with knee osteoarthritis and isolating primary chondrocytes, an in vitro OA model was established by Transwell. Two-dimensionally cultured MSCs (2D culture group) or three-dimensionally cultured MSC microtissues (3D culture group) were co-cultured with primary chondrocytes (Figure A). The expressions of cartilage regeneration-related COL2 and SOX9 genes were significantly up-regulated in chondrocytes of the 3D culture group. And it was verified by Western Blot that COL2 and SOX9 were also highly expressed at the protein level (Figure B). The results indicated that MSC microtissue may be more effective in promoting cartilage regeneration than free cell delivery.

In this study, extensive omics analyses were performed based on in vitro models. Secretome analysis of the co-culture supernatant showed that secretion of GM-CSF was high in the 3D culture group, which plays an important role in cartilage repair, and IL-6R content was low (Panel C). The results showed that MSC microtissue can inhibit inflammation and promote repair compared with free cells. By comparing the differentially expressed genes (DEGs) of various cells in different groups by whole transcriptome RNA sequencing, combined with GO enrichment analysis and KEGG pathway analysis, it was found that in the 3D culture group: MSCs showed a similar process to the early chondrogenic differentiation process gene expression. Chondrocytes showed gene expression more conducive to maintaining articular cartilage-related phenotypes. Moreover, the pathway of cytokine-receptor interaction was more sensitive, and the paracrine interaction between MSCs and chondrocytes was more effective (please refer to the original paper for more details of the analysis). This result further elucidated the potential reason why MSC microtissue is more effective than free cell administration.

[Study Conclusion]

This study proposes that microtissues prepared by 3D large-scale culture process as a stem cell delivery platform can achieve similar efficacy to traditional high-dose delivery methods, while reducing the cell dose and injection frequency required for MSC treatment. Secretome and whole transcriptome analysis showed that microtissue administration was more conducive to promote the communication between MSCs and chondrocytes and contributed to cartilage regeneration through autodifferentiation, anti-inflammatory microenvironment regulation and paracrine effects. The technology of applying 3D FloTrix® Microcarriers is expected to accelerate the application of MSCs in osteoarthritis.

[Study Team]

● This study was jointly completed by Nanjing University, Tsinghua University, and Beijing CytoNiche Biotechnology Co., Ltd.

● Professor Jiang Qing from Nanjing University and Professor Du Ya'nan from Tsinghua University School of Medicine are the corresponding authors of this paper.

● Dr. Wang Bin from Professor Jiang Qing's research group (now working at the First Affiliated Hospital of Zhejiang University) and Dr. Liu Wei, General Manager of Beijing CytoNiche Biotechnology Co., Ltd. are the co-first authors of this paper.

Nanjing University School of Medicine — Professor Jiang Qing

Professor Jiang Qing mainly engage in clinical and basic research in orthopedics and sports medicine, and is the only recipient of the National Outstanding Youth Fund in sports medicine in China.

Professor Jiang Qing is the director of the Osteoarthritis Research Society International (OARSI), vice chairman of the Basic Research Branch of SICOT China Branch, and vice chairman of the International Cartilage Repair Society (ICRS) China Branch.

Professor Jiang Qing have published more than 200 Chinese core papers and more than 100 SCI papers (published in journals including Nature Medicine, Nature Genetic, ACS Nano, etc). In 2007, One of the papers was rated as one of the 100 most influential international papers in China. The total number of citations of the article is 825 times, and the highest single article is cited for 119 times.

Professor Jiang Qing's team has obtained a number of national patents, including 2 patents for 3D printing technology, 1 patent for ankle flexion and extension exerciser, 2 patents for knee joint replacement, 1 patent for repairing articular cartilage damage, etc.

Tsinghua University School of Medicine — Professor Du Ya'nan

He has been dedicated to the innovative exploration in the distinctive interdisciplinary field of "micro-tissue engineering", so as to realize theoretical exploration and technological transformation.

The developed 3D microtissue technology can be used as a new generation of cell-based drug amplification preparation platform and pharmaceutical delivery system, revolutionizing in vitro cell culture and regenerative medicine. At the same time, it can assist in the establishment of biomimetic physiological/pathological models for high-throughput drug screening and pathological mechanism research.

He has published more than 100 high-impact SCI papers (published in journals including Nature Materials, Nature Communications, PNAS, Science Advances, etc.).

He has obtained 16 authorized patents, and Beijing CytoNiche Biotechnology Co., Ltd. was established in 2018 for the transformation, application and commercialization of related microtissue engineering technology patents.

[Materials and Equipment for Research Applications]

3D TableTrix® Microcarriers (Porous Gelatin Microcarriers)|3D FloTrix®vivaSPIN Bioreactors (stirred)|3D FloTrix® miniSPIN Bioreactors (stirred)

Original link:

https://doi.org/10.1016/j.bioactmat.2021.05.029

References:

Expert Consensus on Clinical Drug Treatment of Osteoarthritis

http://www.yixueqianyan.cn/CN/abstract/abstract3673.shtml

[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.

CytoNiche's core product, 3D TableTrix® Microcarrier Tablet (Microcarrier), is an independent innovation and the first pharmaceutical excipient grade microcarrier that can be used for cell drug development. It has obtained the certificate of analysis from relevant authoritative institutions such as National Institutes for Food and Drug Control, and obtained 2 qualifications for pharmaceutical excipients from the National Medical Products Administration (CDE approval registration number: F20210000003, F20210000496). Moreover, the product has obtained the DMF qualification for pharmaceutical excipients from U.S. FDA (DMF: 35481). 

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.