Micropatterning Cells and Spheroids in 3D Hydrogels

Control over cell organization within 3D hydrogels, strategically placing cells in specific locations, holds significant potential for engineering tissues with precise and intricate architectures, as well as for advancing drug testing applications. Although methods for micropatterning cells on 2D substrates are well-established and widely accessible, achieving cell micropatterning within biomimetic 3D hydrogels—mimicking the soft cellular environment—has posed greater challenges, particularly at the micro-scale resolution. Currently, this process relies on sophisticated tools and protocols.

We enable the precise arrangement (‘printing’) of individual cells or cell-spheroids (clusters of cells) with defined sizes within 3D biological hydrogels, such as fibrin and collagen gels. This technique allows for the creation of desired cellular geometries with microscale resolution. As proof of concept, we showcase the capability to micropattern arrays of cells in controlled configurations, such as hexagonal spheroid arrays, and in defined cell-cell spacing. Additionally, we demonstrate the capacity to generate spheroids in predetermined sizes, ranging from individual cells to spheroids of 60, 100, and 200 microns in diameter. Finally, we exhibit control over cell positioning in the Z-dimension (gel thickness-wise) by stacking micropatterned layers together at varying controlled distances.

• A cost-effective and user-friendly method for high-resolution printing of live cells in hydrogels, eliminating the necessity for an advanced bioprinter.
• An effective and precisely controlled method for producing spheroids on 2D surfaces or within 3D biomimetic gels.

• Designing Tissues with Desired Complex Architectures
• Spheroid platforms for drug testing applications

US provisional patent application No. 63/446,531 filed 17-Feb-2023. Bar Ergaz and Ayelet Lesman. Method for micropatterning single cells and spheroids in 3D biological hydrogels.

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