3D cell cultures, such as cell spheroids, are actively used in biology for modeling biological processes, studying intercellular interactions and pharmacological compounds screening and are becoming indispensable objects in cell culture laboratories. There are many methods for producing spheroids, varying in cost and convenience. One of the most handy and affordable is the use of agarose microwells. We have developed approaches to fabricate agarose microwells in standard culture plastic with the assistance of a hobby-grade MSLA 3D printer. The use of 3D printing allows you to customize microwells in a wide range of shapes and sizes and scale the production process from a few spheroids to tens of thousands. We have shown that it is possible to create gel microwells in a dish with a glass bottom, which allows us to easily realize time-lapse confocal microscopy of spheroids, and we have also performed in situ optical clearing in the same dishes to study the spheroid structure. We demonstrated the ability to study the cytotoxicity of various substances and nanoparticles in commonly used 96-well plates. And finally, in this article we describe the difficulties and limitations of our approach and suggest ways for solving them, allowing the reader not only to reproduce it, but also to adapt it to the specific needs of a certain laboratory, using provided 3D models and instructions.

Obesity is a global health threat, which continues to rise in prevalence worldwide, driving increases in related chronic diseases such as type 2 diabetes and cardiovascular conditions. While the sheer scale of the crisis affects over a billion people worldwide, the challenge is compounded for a distinct population: patients with genetically inherent obesity. Gene therapy targeting adipose tissue offers promise for treating obesity, but it requires development of tissue-targeting methods to minimize off-target effects and ensure safety. Our study presents two strategies to restrict transgene expression specifically to adipocytes: engineered adipose-specific promoters and optogenetic regulation. We designed truncated promoter fragments from murine Ucp1, Adipoq, and Fabp4 genes and evaluated their activity during 3T3-L1 preadipocyte differentiation. While all adipocyte-specific promoters exhibited substantially lower activity than the constitutive Cytomegalovirus promoter (10- to 100-fold reduction), they displayed distinct temporal activation patterns: Ucp1 peaked during active differentiation, whereas Adipoq was predominantly active in mature adipocytes. In vivo validation using AAV6/8 vectors confirmed adipose expression of the fluorescent reporter Katushka2S under the Ucp1 promoter in murine interscapular fat. Bioinformatic analysis (MAST/MCAST) of six adipocyte-enriched genes (Fabp4, Adipoq, Ucp1, Lep, Cidec, Sfrp5) identified high-density transcription factor binding site clusters that can be used for the development of new promoters for adipose-specific expression. Furthermore, we implemented the iLight optogenetic system to achieve light-dependent reassembly of split FOXP4 and PRDM16 transcription factors, demonstrating tunable Ucp1 activation with minimal basal expression. These strategies—development of synthetic adipose-specific mini-promoters and light-inducible regulation—may serve as a basis for developing precision metabolic medicine as these approaches allow spatiotemporal control of transgene expression.