Scalable preparation of osteogenic micro‐tissues derived from hESC‐derived immunity‐and‐matrix‐regulatory cells within porous microcarriers in suspension culture

Abstract Bone defects (BDs), a prevalent clinically refractory orthopaedic disease, presently have no effective treatments. Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and serve as potential seed cells for bone tissue engineering for BD treatment. However, the feasibility of using MSCs as seed cells for bone tissue engineering remains unclear. As a result, the critical issue of large‐scale cell‐scaffold preparation remains unresolved. In this study, we demonstrated for the first time that human embryonic stem cell‐derived MSCs, also known as immunity‐and‐matrix‐regulatory cells (IMRCs), could be inoculated into microcarriers to create osteogenic micro‐tissues appropriate for scalable production in 250 mL bioreactor. IMRCs were generally smaller than umbilical cord‐derived MSCs (UCMSCs) and could attach, migrate, proliferate and differentiate within the porous microcarriers, whereas UCMSCs could only attach to the surface of microcarriers. Osteogenic micro‐tissues generated from IMRCs‐seeded microcarriers significantly increased osteocalcin levels after 21 days of differentiation in a bioreactor. Furthermore, the expression levels of osteogenic biomarker genes/proteins such as alkaline phosphatase (ALP), osteocalcin (OCN), runt‐related transcription factor 2 (RUNX2), osteopontin (OPN) and osterix (OSX) were significantly higher than osteogenic micro‐tissues derived from UCMSCs‐seeded microcarriers. Our findings imply that IMRCs could potentially serve as seed cells for the scalable production of osteogenic micro‐tissues for BD treatment.

biofunctional tissues that can integrate and degrade in vivo to fix tissue defects and replace part or all of the functions of lost or failing tissues and organs. 5,6 The selection of seed cells is one of the most significant components in BTE to treat BD. Mesenchymal stem cells (MSCs) are used as a cell population as a source of bone regeneration due to their excellent proliferation and osteogenic ability. 7 However, donor circumstances, the origin of organs or tissues, and techniques of separation, purification and expansion all have a substantial impact on their quality. 8 Human pluripotent stem cell (hPSC)-derived MSCs, when compared to MSCs, provide an alternate source of MSCs due to their comparable or similar phenotypic, immunomodulatory and anti-inflammatory properties. 9 Furthermore, PSC-derived MSCs have significant benefits in terms of differentiation efficiency, purity and cell quality consistency. 10 A previous study reported that human embryonic stem cell (hESC)-derived MSCs, also known as immune and stromal regulatory cells (IMRCs), are promising candidates for regenerative medicine due to their high proliferative capacity and lack of barriers to acquiring primary MSCs. [11][12][13] The high-purity MSCs can be derived from hESCs with the limited passage and good osteogenic ability. 14 However, MSCs alone are insufficient for bone regeneration; generally, they are combined with scaffolds of appropriate shape, size and mechanical properties, in treatment. 15,16 Microcarriers are often considered suitable as spherical scaffolds for cell culture, growth and delivery. 17 Porous microcarriers are widely used in tissue engineering due to their benefits in offering large surface area for cell growth, maintaining differentiated cell phenotypes and facilitating injection into target sites for repair or regeneration. 18,19 In addition, based on their interconnected and open pore structure, microcarriers provide a protective microenvironment for seed cell attachment, proliferation, migration, nutrient exchange and excretion of metabolic wastes. 20,21 Stem cell-based BTE therapies require a minimum of 10 7 -10 9 cells per treatment. To obtain the cell number needed for the clinical application, a stable and controlled expansion system is necessary for the large-scale preparation of stem cells and their derived progenies. 22 Suspension culture technology is a hopeful approach for large-scale expansion and maintaining pluripotency of stem cells. 23 Researchers previously injected human amniotic MSCs into CultiSpher-S microcarriers and attempted osteogenic development in a spinner flask. 24 Porous microcarriers in a suspension culture system were used to grow murine embryonic stem cell (mESC)-derived osteoblasts and chondrocytes cells under feeder layer-free and serum-free conditions. 25,26 However, to date, there is a lack of research on whether hESC-MSCs can be expanded and differentiated into osteogenic cells in a bioreactor.
Therefore, the objectives of the study were to demonstrate the expansion and osteogenic differentiation ability of IMRCs in a bioreactor. It was demonstrated in this work for the first time that scaledup production of IMRCs was possible when combined with porous microcarriers in a bioreactor as well as directly differentiated to form osteogenic micro-tissues.

| Cell monolayer culture in two-dimensional condition
The hESC-derived IMRCs and UCMSCs were obtained following a previous protocol. 13 IMRCs and UCMSCs (passage 4, P4) were seeded on a 6-well tissue culture plate at 1 Â 10 4 cells/cm 2 and expanded in an atmosphere of 5.0% CO 2 at 37 C using medium reported in the previous study. 13 When cells were cultured to 70%-80% confluence, the medium was replaced with osteogenic medium, contain-

| Cell expansion in a bioreactor
3D TableTrix™ (CytoNiche, China) are macroporous microcarriers with a diameter of 100-200 μm. 27 The dry microcarriers (200 mg) were rehydrated in the medium at 37 C until next step. IMRCs and UCMSCs (P4) were seeded onto microcarriers in growth media at densities of 0.5 Â 10 5 , 1.0 Â 10 5 , 1.5 Â 10 5 and 2.0 Â 10 5 cells/mL, respectively, in a 250 mL siliconized bioreactor (Eppendorf, USA) with continuous stirring. Following the cell seeding procedure, the bioreactor culture settings (150-210 rpm) were set to prevent microcarriers from settling to the bottom of bioreactor, pH was set to 7.2, temperature was set at 37 C and dissolved oxygen (DO) tension was set to 40%. The culture was maintained for up to 7 days, and the culture medium was replenished with fresh medium every day, glucose was added daily based on cell density.
The cell-seeded microcarriers were cultured in suspension in the growth medium for 5 days before being replaced with the osteogenic differentiation medium for 21 days cultured in suspension in a bioreactor. The osteogenic differentiation medium was refreshed after every 3 days. The schematic diagram is illustrated in Figure 1.

| Scanning electron microscopy and energy dispersive spectrometer
Cell-laden microcarriers were collected on Days 1, 3, 5 and 7 of expansion phase and Day 21 of osteogenic differentiation phase in a bioreactor. The samples were washed three times with PBS, fixed in 2.5% glutaraldehyde at 4 C overnight, dehydrated in ascending grades of ethanol and air-dried, and examined under scanning electron microscopy (SEM) (Hitachi, Japan). In addition, on Day 21, the cell-laden microcarriers were collected for energy dispersive spectrometer (EDS) analysis (Japan).

| Cell distribution on microcarriers
Cell-laden microcarriers were taken on Day 4 from a bioreactor and fixed in 4.0% paraformaldehyde at 4 C overnight before being permeabilized with 0.1% Triton X-100 for 10 min. The cells were then stained with phalloidin (Invitrogen, USA) and Hoechst 33342 (Invitrogen, USA). Next, they were cut into 30 μm sections, finally, the distribution of cells within the microcarrier was observed using confocal laser scanning microscopy (Zeiss, Germany).  Table S1.

| Transcriptome analysis
The RNA of cells harvested on Day 21 of osteogenic differentiation in 2D was sent to Annoroad Company for mRNA sequencing (RNA-Seq).
Differential genes in the samples were analysed by gene ontology (GO) enrichment analysis.  and anti-rabbit IgG antibody (HRD) (Sigma-Aldrich, USA) at room temperature. Images were obtained using the ChemiDoc XRS + imaging system (Bio-Rad, USA).

| Statistical analysis
Numerical data were represented as mean ± standard deviation. The difference between groups was evaluated by Student's t-test. Statistical significance was *p < 0.05, **p < 0.01 and ***p < 0.001.

| IMRCs possessed faster expansion capability when grown in a 3D culture
Porous microcarriers were utilized to expand MSCs grown under 3D conditions in a bioreactor, and it was found that seeding density affects cell proliferation rate. 27 To explore the optimal seeding density for expansion to increase the utilization of microcarriers, DASbox was seeded at cell densities of 0.5 Â 10 5 , 1.0 Â 10 5 , 1.5 Â 10 5 and 2.0 Â 10 5 cells/mL with TableTrix for 7 days in a bioreactor. The optimal inoculation densities of IMRCs and UCMSCs were 1.5 Â 10 5 and 1 Â 10 5 cells/mL, respectively ( Figure S1). When cells were grown in suspension in a bioreactor, the growth curves showed a typical S type in the expansion phase, and the maximum cell density of IMRCs and UCMSCs were 9.99 Â 10 5 and 5.99 Â 10 5 cells/mL, respectively ( Figure 3A), indicating that the higher density of IMRCs can be achieved than UCMSCs when they were cultured in bioreactors with optimal seed densities. As a result, as compared to UCMSCs culture, the residual glucose concentrations for IMRCs with greater glucose consumption rates over the whole suspension process were slightly  Figure 3E).
Conclusively, a large number of IMRCs with high cell viability could be generated in a bioreactor culture system with porous microcarriers.

| Distribution of IMRCs in cell-loaded porous microcarriers
Porous scaffolds feature a high density of interconnected 3D porous structures, making them more conducive to cell migration and nutrient supply than hydrogels. 30,31 The average inner pore size of TableTrix porous microcarriers was 20 ± 5.7 μm. 27 SEM ( Figure 4A In principle, they can grow in microcarrier holes. Immunofluorescence staining of cells was observed using confocal microscopy and revealed that IMRCs and UCMSCs were distributed on the surface of the porous microcarriers ( Figure 4C). However, the section staining results showed that IMRCs were distributed in the centre and at the edges of the microcarriers, while UCMSCs were just distributed at the edges ( Figure 4D). Therefore, the results presented here demonstrated that only IMRCs can grow and migrate in and out of porous microcarriers in a bioreactor.  Figure 5E). Therefore, these results demonstrated that cells were seeded onto microcarriers to form osteo-microtissue after osteogenic differentiation. Moreover, the osteogenic capacity of IMRCs-seeded microcarriers formed osteogenic micro-tissues was superior to the osteogenic microtissues formed from UCMSC-seeded microcarriers.

| DISCUSSION
MSCs have a wide range of application prospects in tissue engineering. Like MSCs, IMRCs have immune privileges that allow them to evade allogeneic immune responses. In our previous study, we demonstrated the potential of IMRCs, in which a soft agar assay was performed, and no colonies were formed. 13 In addition, IMRCs could not form any tumour in immunodeficient mice after injection in vivo. 13 So, it is concluded that the IMRCs have promising application prospects in BTE. Moreover, it is well known that the expansion and osteogenic abilities of stem cells are crucial for the application of BTE, 32  In this study, the area utilization rate of microcarriers was further enhanced because the diameter of IMRCs was lower than that of UCMSCs. It can easily be scaled up to produce as many cells as necessary for transplantation.
To treat bone injury, osteogenic micro-tissues should be produced on a large scale. Only a few researchers have explored the use of bioreactors in the tissue engineering of bone constructs from MSCs. Previous studies found that MSCs can combine CMPs particles to form osteogenic micro-tissues in spinner flask. 21,35 However, this was the first time IMRCs were differentiated to osteoblasts in a bioreactor using TableTrix microcarriers. It might be used as cell-scaffold composites for possible BD treatment.
However, there were some limitations in this study. For example, the presence of dead cells in osteogenic micro-tissues needs to be further investigated and a detection method to quantify the amount of dead cells in the osteogenic micro-tissues is required. Moreover, the exact mechanism underlying this discrepancy between the osteogenic potential of IMRCs and UCMSCs is currently unknown. It is possible that several factors, such as culture conditions (including oxygen tension and mechanical stimulation), microcarrier materials and reaction systems, may affect cell proliferation and differentiation. 36,37 Therefore, further studies are required to optimize and explore the culture parameters in a bioreactor.

| CONCLUSION
To summarize, we investigated the proliferative and osteogenic potential of IMRCs and UCMSCs. The results showed that IMRCs had a greater proliferative and osteogenic capacity between 2D and 3D cultures. Additionally, the porous microcarriers were adequate for promoting the proliferation and osteogenic differentiation of IMRCs compared with UCMSCs, which can differentiate into osteogenic micro-tissues, allowing for the manufacture of millimetre-sized osteogenic micro-structures in a bioreactor. In conclusion, these findings suggest that IMRCs might be employed as seed cells in BTE.