Engineering Human Mesenchymal Bodies in a Novel 3D-Printed Microchannel Bioreactor for Extracellular Vesicle Biogenesis

Abstract

Human Mesenchymal Stem Cells (hMSCs) and their derived products hold potential in tissue engineering and as therapeutics in a wide range of diseases. hMSCs possess the ability to aggregate into "spheroids", which has been used as a preconditioning technique to enhance their therapeutic potential by upregulating stemness, immunomodulatory capacity, and anti-inflammatory and pro-angiogenic secretome. Few studies have investigated the impact on hMSC aggregate properties stemming from dynamic and static aggregation techniques. hMSCs' main mechanistic mode of action occur through their secretome, including extracellular vesicles (EVs)/exosomes, which contain therapeutically relevant proteins and nucleic acids. In this study, a 3D printed microchannel bioreactor was developed to dynamically form hMSC spheroids and promote hMSC condensation. In particular, the manner in which dynamic microenvironment conditions alter hMSC properties and EV biogenesis in relation to static cultures was assessed. Dynamic aggregation was found to promote autophagy activity, alter metabolism toward glycolysis, and promote exosome/EV production. This study advances our knowledge on a commonly used preconditioning technique that could be beneficial in wound healing, tissue regeneration, and autoimmune disorders.

Keywords: 3-D aggregates; extracellular vesicle biogenesis; human mesenchymal stem cells; microchannel; wave motion.

Figure 2

3-D printed microchannel bioreactor and wave motion fluid dynamics. Schematics detailing the preparation of hMSC aggregates. Initial draft of design is fabricated in SOLIDWORKS. Design is 3D-printed using DREMEL^® DigiLab 3D Printer 3D45. PDMS is added to the 3D mold forming the 6-well insert. An additional layer of PDMS is added to the microchannels smoothing out the surface. Channels are sterilized by soaking them in ethanol overnight under ultraviolet light. Channels are washed twice with PBS and then incubated with 5% bovine serum albumin (BSA) solution. Channels are seeded with hMSCs and rocked overnight at 20 RPM and 8° forming the aggregates. Scale bar: 200 µm.

Figure 3

HMSC aggregate morphology and characterizations. (A) Aggregate formation at different seeding density. Scale bar: 200 µm. (B) Aggregate morphology for dynamic and static culture. Scale bar: 200 µm. (C) hMSC aggregate cell number change over time. (D) hMSC aggregate size change over time. hMSC aggregate size and cell number decrease throughout culture period. Dynamically cultured aggregates decrease size at a faster rate than their static counterparts. Cell number remains higher in dynamic aggregates through six days of culture compared to static culture. * indicates p < 0.05.

Figure 1

3-D printed microchannel design. Multiple design iterations of the microchannel reactor. (A) First design iteration in SOLIDWORKS and 3D print filled with PDMS. (B) Second design iteration in SOLIDWORKS and 3D print filled with PDMS. (C) hMSCs attaching to bottom of PDMS channel. Scale bar: 200 µm. (D) Final design iteration of 3D mold and PDMS cast.

Figure 4

Characterizations of the isolated EVs by nanoparticle tracking analysis (NTA) and protein assay. Dynamic aggregation increases hMSC-EV secretion. (A) NTA analysis of EV size distributions. (B) Mean EV size; (C) Dynamic aggregation increases EV secretion roughly 5.2-fold over static aggregation and 2.7-fold over the 2D control. (D) Protein content normalized to EV number. (E) Transmission electron microscopy image for the isolated EVs of dynamic 3D culture. Scale bar: 60 nm. * indicates p < 0.05.

Figure 5

Expression of metabolic markers and EV biogenesis markers. mRNA expression of EV biogenesis markers in the cells were determined by RT-PCR. (A) Genes related to cellular metabolism; (B) Genes related to autophagy; (C) EV biogenesis markers-ESCRT dependent; (D) EV biogenesis markers-ESCRT independent. N = 3. * indicates p < 0.05; ** indicates p < 0.01, *** indicates p < 0.001.