A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss

Abstract

Spheroid cultures have been often used to simulate and understand in situ biological occurrences with potential to be further applied to therapeutic approaches, such as cell transplantation. However, traditional lab-scale techniques hardly reached the needed large scale production of cell spheroids, thus limiting their versatility in many biomedical fields. Microscale technologies have rapidly improved in the last decade, and contributed to the large scale production of cell spheroids with high controllability and reproducibility. Nonetheless, the existing microwell culture platforms are problematic due to unwanted cellular adhesion to the substrate as well as due to substantial amounts of cell loss. In this study, we have developed a novel configuration of cylindrical type polyethylene glycol (PEG) hydrogel microwells featuring inverted-pyramidal openings (iPO). Highly refined microstructures of our novel microwell could be fabricated by our optimized micro-electro mechanical system protocols consisting of a silicon (Si) wet/dry etching, Si-to-polydimethylsiloxane substrate bonding, and the established soft-lithography techniques. The iPO, the PEG hydrogel, and the cylindrical geometry of our microwell successfully (1) avoided inefficient washing steps after cell seeding, (2) achieved the complete resistance to cellular adhesion on the microwell substrate, and (3) made all seeded cells readily gathered and jam-packed to form cell spheroids with uniform size, respectively. The maximal sizes of cell spheroids were confined to below 200 μm according to the size of microwells used in this study. The efficiency testing for cell spheroid formation was conducted in comparison with other types of microwells that have been often used in the fields. The results showed that our novel microwell platform effectively reached almost zero percent of cell loss while mass-producing human mesenchymal stem cell spheroids with highly precise control over spheroid's size and cell number. We believe that this study could deliver an effective method to extend the practical usability of cell spheroids in a variety of biomedical applications.

Simple In-House Fabrication of Microwells for Generating Uniform Hepatic Multicellular Cancer Aggregates and Discovering Novel Therapeutics

“…That is, in the 2D condition, cells are directly exposed to the drug during incubation; by contrast, the densely packed cell–cell junctions in MCAs piled into multiple layers and reduced the penetration of toxicants, which explains why only cells located at the periphery appeared to be damaged (Figure 4A). In fact, this resemblance of tissue structures was found to increase the resistance of anticancer drugs in 3D MCAs, as reported elsewhere [15]. Similarly, Breslin et al used the HER2-positive breast cancer cell line as a 3D MCA model (BT474, HCC1954 and EFM192A) and found that cells cultured by the 3D method showed more drug resistance to anticancer agents than cells cultured in 2D conditions [37].…”

“…However, given that M −3@15 exhibited a larger aspect ratio, which tends to disrupt cells during medium exchange operations, only a portion of the medium was exchanged, resulting in cells remaining in the gap of the microwell that may promote uneven MCA distribution. Recent reports demonstrated that controlling the density of the microwells by reducing the gaps between them can enhance the formation of regular uniform MCAs and save the precious sample from being depleted during the medium exchange procedure [15,36]. Although M −3@15 was not selected for subsequent functional assays owing to the issue presented, this outcome highlights the potential improvements that could be further implemented to empower the applicability of the presented method.…”

“…Various approaches have been proposed to generate MCAs, e.g., nonadherent plates [11], spinner culture [12], hanging drop [13] and microfabrication techniques [14,15]. The performance criteria generally depend on trade-offs in uniformity, throughput, cost and ease of usability in producing MCAs, as well as the integration of subsequent applications (reviewed by Ahn et al [16]. )…”

Efficient scalable production of therapeutic microvesicles derived from human mesenchymal stem cells

“…Large-scale formation of hMSC-spheroids was accomplished using a PEG hydrogel microwell array modified from previous studies 20 – 22 . Our custom-engineered microwell array was composed of cylindrical microwells with inverted-pyramidal openings (Fig.…”

“…Therefore, in this study, we hypothesized that the production of therapeutic MVs could be amplified by enhancing the biological functions of MSC-aggregates as dynamic 3D-culture progressed. First, to achieve the large-scale formation of size-controlled hMSC-spheroids in a reproducible manner, we adopted and modified a PEG hydrogel microwell array platform with an inverted-pyramidal opening structurally adjoining each cylindrical microwell 22 . The unique features of our modified PEG microwell array completely prevented cell loss during the large-scale formation of hMSC-spheroids, resulting in significant cost-saving through reduced wastes of expensive cell materials.…”