Large-scale Production of Stem Cells Utilizing Microcarriers

Editor's Notes

• #5: Human stem cells, including pluripotent, embryonic and mesenchymal, stem cells play pivotal roles in cell-based therapies. Additionally, some bone, skin and corneal diseases or injuries can be treated by grafting tissues that are derived from or maintained by stem cells. These therapies have also been shown to be safe and effective. MSCs have therapeutic potential for treating heart [18], diabetes [19,20], gastrointestinal [21], liver [22], kidney [17], immune [23,24] and neurodegenerative diseases [[25], [26], [27], [28]] as well as to regenerate bone [[29], [30], [31]] and cartilage [10,32,33]. On the other hand, PSCs including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) currently have limited utility in cell therapy trials due to risk of tumorigenicity [34]. hESCs have been tested for treatment of spinal cord injury [[35], [36], [37]], retinal pigment epithelial (RPE) transplantation [[38], [39], [40]], rare diseases [41] and treatment of stargardt's macular dystrophy (SMD) [42]. hiPSCs are also used as models for studying diseases such as Parkinson's [25], Alzheimer's [43,44], juvenile onset [45], type I diabetes mellitus [46] and Duchenne type muscular dystrophy [
• #6: Cell-based therapy and tissue regeneration approaches for human diseases demand a larger number of cells. Stem cells have emerged as promising therapeutic agents owing to their promising traits such as differentiation capacity for treatment of various diseases. Mesenchymal Stem Cells (MSCs) and Pluripotent Stem Cells (PSCs) are two distinct types of stem cells which have been successfully used in human clinical trials
• #7: The current hallmark of SARS-CoV-2 pathogenesis is the cytokine storm in the lung. Virally-triggered acute cytokine release of GSCF, IP10, MCP1, MIP1A, IL-2, IL-6, IL-7, and TNF results in pulmonary edema, dysfunction of air-exchange, acute respiratory distress syndrome (ARDS), and acute cardiac injury, and leading to death
• #8: . Probably, MSC therapy can prevent the storm release of cytokines by the immune system and promote endogenous repair by reparative properties of the stem cells
• #9: Conceptual illustration of a technology S-curve showing the evolution of expansion technologies used in cell therapy manufacture. This curve demonstrates the performance of each technology in terms of billion cells achieved per lot (when using the maximum number of units per lot) against R&D effort/investment. The x-axis represents qualitatively the R&D effort required for a company currently using T-flasks to change to other cell expansion technologies. Cost values are calculated based on the direct costs (material, labor, QC testing) and indirect costs (equipment depreciation only) of the cell expansion process and assuming overheads are spread over 10 lots/year for all scenarios.  Conventional monolayer culture of cells could lead to alteration of cell specific ECM secretion, loss of specific morphology and phenotype during passaging [3]. Scaled up manufacturing of the cells using microcarriers in suspension cultures is a promising tool to minimize the limitations of monolayer culture. Three-dimensional (3D) culture of cells allows cells to retain phenotypes and prevent dedifferentiation, during mechanically stimulation
• #10:  Different bioreactor systems used in 3D cell culture. (A) Wave bioreactor, (B) Stirred Tank Bioreactor, (C) Hollow Fiber Bioreactor, (D) Rotating Wall Vessel Bioreactor, (E) Packed-bed bioreactor, (F) Roller Bottle Bioreactor. ells in the order of billions to trillions are required for most of cell therapies. Stem cells isolated from donors are initially expanded in 2D culture flasks. Hereafter, the expansion from millions to hundreds of billions in quantity can be realized with microcarriers in bioreactors. Bioreactors play a crucial role in expanding cells in a quasi-physiologic manner, as cells reside and grow in 3D environment in-vitro. However, some major issues such as the exchange of nutrients/metabolites gradients, difficulties in recovery of cells after expansion and induced shear stress caused by moving culture media or rotating shaft must be overcome
• #11: G) Schematic of the BioLevitator as a bench 3D cell culture equipment [52]. (H) Global Eukaryotic Microcarrier (GEM) microcarrier with its especial features. For 3D cell culture experiments in laboratory scale, automated benchtop platforms have been developed. BioLevitator™ is a small 3D cell culture system that provides all the functions of a bioreactor placed into an incubator. Lin et al. [52] used this system to study expansion and differentiation of human adipose derived MSCs (hASCs) on two commercial microcarriers (i.e., magnetic microcarrier GEM and cytode×3). Global Eukaryotic Microcarrier (GEM) is a paramagnetic microcarrier composed of an alginate core where small paramagnetic particles are dispersed in. It is also available with different coatings (gelatin, collagen, fibronectin, etc.). Its magnetic properties facilitate easy control during media exchange, harvesting and assay washes [53]. BioLevitator allows tuning the agitation speed and rotating periods in a programmable manner (Fig. 2G–H).
• #12: (A) Different properties of microcarriers that can determine stem cells attachment, proliferation and differentiation on them. (B) Microcarriers could be classified based on i) material, ii) the substance that cells grow either solid or liquid, iii) surface topography of carrier and iv) geometry.   Their properties differ in terms of material composition, size, shape, morphology, surface coating/charge, functional groups, and stiffnes
• #13: C, D and E shows the main parts of the current articles where we first introduce the commercially available microcarriers and their characteristics (C). Then the microcarriers with tissue engineering capabilities, including different types and recent advances are discussed (D). Lastly, the final part covers the stimulus responsive MCs as various stimuli such as temperature, pH, light, chemicals and electric or magnetic field could be used to trigger a specific cell behaviour (E). 
• #14:  Three typical strategies for cell delivery and tissue regeneration. i) cell seeded 3D porous scaffolds, ii) cell-mixed hydrogel and iii) Cell-laden microcarrier. B) Three distinct types of microcarrier that are used for controlled encapsulation and release. Image is adopted from Ref. [88]. C) Schematic of linear and star shaped PLLA nanofibrous microspheres and nanofibrous hollow microsphere. i: SEM image of a nanofibrous microsphere, showing the nanofibrous architecture on the microsphere surface. ii: SEM image of a nanofibrous hollow microsphere, showing the nanofibrous architecture and a hole of approximately 20 μm on the microsphere shell. iii: A 2D cross-section confocal image of the nanofibrous hollow microspheres. iv: A high-magnification image of the microsphere in ii, showing the nanofibers, which have an average diameter of about 160 nm. Images are reproduced from Ref. [91]. D) Dissipative particle dynamics simulations and SEM images of 16-arm star shaped PLLA. 
• #17: To control stem cells differentiation fate, specific features could be manipulated to design fate dependent carriers. For example, by altering surface functional groups, e.g. adding NH2 groups MSC proliferation, spreading and osteogenic commitment will promote [67,136], while introduction of COOH groups lowers MSC spreading and enhances chondrogenesis [136,137]. Rigid surfaces (stiffness of 34 kPa) promoted spindle-like shape and osteogenic differentiation of MSCs [138], while softer substrates (i.e. 1 kPa) encouraged chondrogenic, adipogenic or neuronal differentiation [68,137]. Intermediate stiffness promoted muscular lineage [[139], [140], [141]]. For MSCs, surface of microcarriers coated with large amounts of ECM protein facilitates spreading, proliferation and osteogenic differentiation of MSCs (through activation of RhoA/ROCK signalling) [74,136,138], while softer carriers with small ECM coating enhances adipogenesis or chondrogenesis [142]. For PSCs, microcarriers with intermediate stiffness covered with large amount of ECM differentiate to mesodermal linages [143,144], while smaller ECM distribution directs ectodermal differentiation [145]. For similar carriers, restricted ECM coating, supports endodermal commitment (Fig. 8) [63]. 
• #18: MCs with higher mechanical stability in tissue engineering, studies have focused on the selection of synthetic biomaterial with specific chemical properties and morphology to direct stem cell fate. ceramics containing calcium ions (Ca2+) and phosphate anions have been used in the fabrication of MCs to enhance osteogenic differentiation and proliferation of bone cells
• #19: Zeolitic imidazolate frameworks (ZIFs), a subclass of metal–organic frameworks, are hybrid materials constructed by tetrahedral building blocks in which metal ions (M = Co and Zn) connect to nitrogen atoms of imidazole-derived ligands. 
• #20: Light microscopy images Characterization of PDA MCs and ZIF8 MCs. (A) ATR-FTIR spectra of PS beads, PDA MCs and ZIF8 MCs. (B) XRD patterns of PS beads, PDA MCs, ZIF8 MCs, and ZIF8 database. (C) Energy dispersive spectra (EDX) of ZIF8 MCs. (D) Water contact angle and surface free energy measurement of TCP, PDA-TCP and ZIF8-TCP with diiodomethane, water and glycerol using Van Oss method. Each sample was assessed in three replicates.
• #21: SEM images of PS beads (A1–A3), PDA MCs (B1–B3), ZIF8 MCs (C1–C3).
• #22:  PS beads: (A1) day 1, (A2) day 3, (A3) day 7. PDA MC: (B1) day 1, (B2) day 3, (B3) day 7. ZIF8 MC: (C1) day 1, (C2) day 3, (C3) day 7. Star-Plus MCs: (D1) day 1, (D2) day 3, (D3) day 7. The images of PS beads and PDA MCs in different days of culture indicate cell proliferation enhanced by PDA coating. Using ZIF8 thin film layer in the fabrication of
• #23: Fluorescent microscopy images of hADSCs attachment and growth on MCs under static condition
• #24: (A) Total viable cell density obtained by 3D expansion of cells on PS MCs, PDA MCs, ZIF8 MCs, and Star-Plus MCs after 7 days of culture. ZIF8 MCs supported a high level of ADSCs (5.8 fold expansion). (B) Analysis of cell attachment and growth on the surface of PS beads, PDA MCs, ZIF8 MCs, and Star-Plus MCs using MTS assay. PDA and ZIF8 coating improve cell attachment and growth of PS beads with a significant difference. Multipotency assay of harvested hADSCs from (C) PDA MCs (osteoblasts, left panel) (adipocytes, right panel) and (D) (osteoblasts, left panel) (adipocytes, right panel). PDA and ZIF8 MCs retained their differential potential under dynamic conditions
• #25: ZIF8 thin film layer coating could effectively promote surface property of PS beads such as wettability, roughness, surface charge, and surface free energy to stimulate cell attachment and growth in both dynamic and static cell culture conditions. The ZIF8 MCs were biocompatible and maintained their differentiate potential in 3D cell culture. They achieved greater cell growth and cell yield compared with other tested MCs. ZIF8 MCs were fabricated by modifying PS beads with ZIF8/PDA/PEI at room temperature without using any toxic or organic solvents. A ZIF8 crystal layer was formed on the surfaces of MCs, possessing the mechanical properties necessary to support cells in bioreactors. The proposed method for fabrication of MCs is non-toxic, cost-effective, and straightforward that may be adopted as a generalized method for converting non-wetting beads to bioactive and tunable MCs with high cell adhesion and viability.