large-scale-cell-production-based-on-gmp-grade-dissolvable-porous

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...

Beijing CytoNiche Biotechnology Co. Ltd.

Mesenchymal stem cell (MSC)-based therapy to combat diabetic-associated metabolic disorders is hindered by impoverished cell survival and limited therapeutic effects under high glucose stress. Here, we genetically engineered MSCs with Exendin-4 (MSC-Ex-4), a glucagon-like peptide-1 (GLP-1) analog, and demonstrated their boosted cellular functions and antidiabetic efficacy in the type 2 diabetes mellitus (T2DM) mouse model. Mechanistically, MSC-Ex-4 achieved self-augmentation and improved survival under high glucose stress via autocrine activation of the GLP-1R-mediated AMPK signaling pathway. Meanwhile, MSC-Ex-4-secreted Exendin-4 suppressed senescence and apoptosis of pancreatic β cells through endocrine effects, while MSC-Ex-4-secreted bioactive factors (e.g., IGFBP2 and APOM) paracrinely augmented insulin sensitivity and decreased lipid accumulation in hepatocytes through PI3K-Akt activation. Furthermore, we encapsulated MSC-Ex-4 in 3D gelatin microscaffolds for single-dose administration to extend the therapeutic effect for 3 months. Together, our findings provide mechanistic insights into Exendin-4-mediated MSCs self-persistence and antidiabetic activity that offer more effective MSC-based therapy for T2DM.

Exendin-4 gene modification and microscaffold encapsulation promote self-persistence and antidiabetic activity of MSCs.

Mesenchymal stem cells (MSCs) can be effective in alleviating the progression of osteoarthritis (OA). However, low MSC retention and survival at the injection site frequently require high doses of cells and/or repeated injections, which are not economically viable and create additional risks of complications. In this study, we produced MSC-laden microcarriers in spinner flask culture as cell delivery vehicles. These microcarriers containing a low initial dose of MSCs administered through a single injection in a rat anterior cruciate ligament (ACL) transection model of OA achieved similar reparative effects as repeated high doses of MSCs, as evaluated through imaging and histological analyses. Mechanistic investigations were conducted using a co-culture model involving human primary chondrocytes grown in monolayer, together with MSCs grown either within 3D constructs or as a monolayer. Co-culture supernatants subjected to secretome analysis showed significant decrease of inflammatory factors in the 3D group. RNA-seq of co-cultured MSCs and chondrocytes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed processes relating to early chondrogenesis and increased extracellular matrix interactions in MSCs of the 3D group, as well as phenotypic maintenance in the co-cultured chondrocytes. The cell delivery platform we investigated may be effective in reducing the cell dose and injection frequency required for therapeutic applications.

A low dose cell therapy system for treating osteoarthritis:  study and  mechanistic investigations.

The emerging field of cultured meat faces several technical hurdles, including the scale-up production of quality muscle and adipose progenitor cells, and the differentiation and bioengineering of these cellular materials into large, meat-like tissue. Here, we present edible, 3D porous gelatin micro-carriers (PoGelat-MCs), as efficient cell expansion scaffolds, as well as modular tissue-engineering building blocks for lab-grown meat. PoGelat-MC culture in spinner flasks, not only facilitated the scalable expansion of porcine skeletal muscle satellite cells and murine myoblasts, but also triggered their spontaneous myogenesis, in the absence of myogenic reagents. Using 3D-printed mold and transglutaminase, we bio-assembled pork muscle micro-tissues into centimeter-scale meatballs, which exhibited similar mechanical property and higher protein content compared to conventional ground pork meatballs. PoGelat-MCs also supported the expansion and differentiation of 3T3L1 murine pre-adipocytes into mature adipose micro-tissues, which could be used as modular assembly unit for engineered fat-containing meat products. Together, our results highlight PoGelat-MCs, in combination with dynamic bioreactors, as a scalable culture system to produce large quantity of highly-viable muscle and fat micro-tissues, which could be further bio-assembled into ground meat analogues.

Engineered meatballs via scalable skeletal muscle cell expansion and modular micro-tissue assembly using porous gelatin micro-carriers.

Efficient and large-scale expansion of mesenchymal stem/stromal cells (MSCs) has always been a formidable challenge to researchers in cell-based therapies and regenerative medicine. To reconcile major drawbacks of 2D planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on GMP-grade microcarrier for culture of umbilical cord-mesenchymal stem/stromal cells (UCMSCs), in accordance with the criteria of stem cell bank. ACISCP system is a fully closed system, which employs different models of vivaSPIN bioreactors (CytoNiche Biotech, China) for scale-up cell culture and vivaPREP (CytoNiche Biotech, China) for automated cell harvesting and cell dosage preparation. To realize industrial scale expansion of UCMSCs, a three-stage expansion was conducted with 1 L, 5 and 15 L vivaSPIN bioreactors. Using 3D TableTrix and ACISCP system, we inoculated 1.5 × 10 of UCMSCs into 1 L vivaSPIN bioreactor and finally scaled to two 15 L bioreactor. A final yield of 2.09 × 10 cells with an overall expansion factor of 1975 within 13 days. The cells were harvested, concentrated, washed and prepared automatically with vivaPREP. The entire process was realized with ACISCP platform and was totally enclosed. Critical quality attributes (CQA) assessments and release tests of MSCs, including sterility, safety, purity, viability, identity, stability and potency were performed accordingly. The quality of cells harvested from 3D culture on the ACISCP and conventional 2D planar culture counterpart has no significant difference. This study provides a bioprocess engineering platform, harnessing GMP-grade 3D TableTrix microcarriers and ACISCP to achieve industrial-scale manufacturing of clinical-grade hMSCs.

Yuanyuan Zhang

Beijing CytoNiche Biotechnology Co. Ltd.

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers