A2576

Agarose micro-wall arrays were fabricated on a cell culture surface as previously described [34 ]. Briefly, the surface of the PDMS micro-mold was cleaned with mending tape (MP-18) (Sumitomo 3M, Tokyo). After rinsed with ethanol and deionized water, the PDMS micro-mold was placed on a PLL coated glass bottom dish (D11141H) (Matsunami Glass Ind., Osaka). The mold and dish were placed in a vacuum desiccator connected to a vacuum line and degassed at a gauge pressure of -98 kPa for 1 hour. Immediately after degassing, the mold and dish were removed from the desiccator and 2w/v% agarose aqueous solution (A2576) (Sigma-Aldrich, St. Louis, MO) heated to boiling in a microwave oven was poured into the hole on the PDMS micro-mold by a micro-pipette. It was placed on a plate heater with a surface temperature of 50°C until the agarose solution was guided into the PDMS micro-mold by negative pressure and completely filled the space inside. The mold and dish were then cooled at 4°C for 30 min to induce the gelation of agarose. The gelated agarose was dehydrated in a drying oven (EO-300B) (ASONE) at 70°C for 2 d. Peeling off the PDMS micro-mold, the dehydrated agarose patterns remained on the surface of the glass bottom dish. The surface of micro-wall was monitored with a three-dimensional laser scanning confocal microscope (VK-8710) (Keyence, Osaka).

1.5% w/w sodium alginate solution (NaAlg; Wako, 194–13321) and 150 mM calcium chloride solution (CaCl₂; Wako, 039–00475) were used during fabrication of hydrogel microsprings, while propylene glycol alginate (PGAlg; Wako, 165–17415) was used as an inner solution during fabrication of tubular microsprings. To produce heterogeneous core-shell hydrogel microsprings, 3% w/w agarose solution (SIGMA-ALDRICH, A2576) with a low melting point, a mixture composed of 5% v/v magnetic fluid (Ferrow Tec, EMG707) and 2.85% w/w NaAlg, and 2% w/w bovine dermal type-I collagen solution (IAC-50, KOKEN) containing HepG2 cells with a concentration of 1.0 × 10⁸ cells/mL were used as core materials. To remove calcium alginate shells, a solution containing 200 μg/mL of alginate lyase (SIGMA, A1603) in phosphate-buffered saline (×10 PBS(−), WAKO, 163–25265, was diluted by sterilized water) was utilized. To form the collagen-core microspring encapsulating HepG2 cells, a solution mixture containing 1.5% w/w NaAlg and 145 mM NaCl (Wako, 191–01665) was used as the shell flow.

To securely immobilize and precisely position non-anesthetized non-paralyzed zebrafish larvae for extended light-sheet imaging sessions, we devised a process through which larvae can be embedded in a cylinder of 1.3% ultra-low gelling temperature agarose (which solidifies at 25 °C; A2576, Sigma) surrounded by shell of 2% low gelling temperature agarose (which solidifies at 55 °C; A0701, Sigma). The ultra-low gelling temperature core allows larvae to be safely added to the agarose while in a liquid state without being exposed to excessive temperatures. The more rigid low gelling temperature agar shell strengthens and supports the inner core, allowing it to be inserted into the glass capillary and ensuring that the larvae are fully immobilized. Embedding is accomplished by first transferring larvae into a solution of liquid 1.3% ultra-low gelling temperature agarose, which is then poured into a 20 mL syringe. The 20 mL syringe is then inserted into a 60 mL syringe filled with 2 % low gelling temperature agarose. The 20 mL syringe is capped with an 18-gauge stainless steel needle and the 60 mL syringe is capped with a 16-gauge stainless steel needle. We then simultaneously extrude both agarose solutions into a room temperature bath containing E3 medium, allowing the agar to rapidly solidify.