Thiolated hyaluronic acid

Immune enhanced tumor organoids (iTOs) were created by encapsulating ~ 10 × 10⁶ tumor cells and ~ 30 × 10⁶ immune cells counted with a Nucleocount NC-200 (Chemometec, Denmark) per 1 mL of a 3:1 mixture of 3 mg/mL methacrylated collagen (Advanced Biomatrix, USA) suspended in 20 mM acetic acid (Advanced Biomatrix, USA) and 1 mg/mL thiolated hyaluronic acid (Advanced Biomatrix, USA) suspended in 0.1% w/v irgacure photo-initiator (Advanced Biomatrix, USA) in autoclaved DO water. The hydrogel mixture was neutralized with 95ul of Neutralization Solution (Advanced Biomatrix, USA) per 1 mL of 3 mg/mL collagen utilized. This leads to a final concentration of 2.25 mg/mL collagen and 0.25 mg/mL collagen in the organoid. 10µL of the mixture (equal to 1 × 10⁵ 4T1, 3 × 10⁵ immune or 4 × 10⁵ combined cells) was pipetted into individual wells in a 48 well plate (Falcon, USA) coated with 120 µL PDMS (DOW Chemical, USA) to prevent cell outgrowth outside the organoids. The hydrogel mixture was exposed to 13.2 J/m³ UVA radiation for two and a half seconds to initiate the crosslinking (gelation) of the hydrogel to form the individual organoids. Pervious work with this hydrogel system has demonstrated that no phenotypic and viability changes in cells are observed^{19 (link),26 (link),38 (link)}. Control organoids were also created with either the immune cells or the tumor cells only.