Engineering spheroids potentiating cell-cell and cell-ECM interactions by self-assembly of stem cell microlayer

Area of Science:

• Biomaterials Engineering
• Stem Cell Biology
• Tissue Engineering

Background:

• Current stem cell spheroid fabrication methods have limitations in cell viability, throughput, and recapitulating the natural microenvironment.
• Existing techniques often rely on trypsinized, suspended cells, which can compromise cell health and function.

Purpose of the Study:

• To develop an improved system for engineering 3D stem cell spheroids using self-assembly of micro-scale monolayers.
• To investigate the influence of micropattern geometry and cellular mechanics on spheroid formation and quality.

Main Methods:

• Engineered synthetic hydrogels with chemically formed square and circle micropatterns (200 μm).
• Cultured human nasal turbinate-derived mesenchymal stem cells (hTMSCs) to form monolayers on micropatterns.
• Utilized thermally controlled hydrogel expansion and contraction for rapid cell layer delamination and spheroid self-assembly.
• Analyzed spheroidization efficiency, cell viability, gene and protein expression, and differentiation markers.

Main Results:

• Rapid spheroid formation (>90% viability) achieved by decreasing temperature, detaching monolayers from micropatterns.
• Square micropatterns promoted more efficient spheroidization than circular patterns, linked to stress concentration.
• Spheroids from monolayers exhibited higher expression of extracellular matrix proteins, stemness markers (Oct4, Sox2, Nanog), and tri-lineage differentiation markers compared to conventional methods.
• Results were validated using adipose-derived mesenchymal stem cells (ADSCs).

Conclusions:

• The novel micropattern-guided self-assembly technique enables efficient, high-viability 3D stem cell spheroid generation.
• This method enhances key stem cell characteristics and differentiation potential, outperforming traditional low-attachment plate methods.
• The technique shows significant promise for regenerative medicine, offering improved 3D cell culture and therapeutic cell delivery strategies.