Hydrogels can facilitate nucleus pulposus (NP) regeneration, either for clinical application or research into mechanisms of regeneration. However, many different hydrogels and culture conditions for human degenerated NP have been employed, making literature data difficult to compare. Therefore, we compared six different hydrogels of natural polymers and investigated the role of serum in the medium and of osmolarity during expansion or redifferentiation in an attempt to provide comparators for future studies. Human NP cells of Thompson grade III discs were cultured in alginate, agarose, fibrin, type II collagen, gelatin methacryloyl (gelMA), and hyaluronic acid-poly(ethylene glycol) hydrogels. Medium containing fetal bovine serum and a serum-free (SF) medium were compared in agarose, gelMA, and type II collagen hydrogels. Isolation and expansion of NP cells in low compared to high osmolarity medium were performed before culture in agarose and type II collagen hydrogels in media of varying osmolarity. NP cells in agarose produced the highest amounts of proteoglycans, followed by cells in type II collagen hydrogels. The absence of serum reduced the total amount of proteoglycans produced by the cells, although incorporation efficiency was higher in type II collagen hydrogels in the absence than in the presence of serum. Isolation and expansion of NP cells in high osmolarity medium improved proteoglycan production during culture in hydrogels, but variation in osmolarity during redifferentiation did not have any effect. Agarose hydrogels seem to be the best option for in vitro culture of human NP cells, but for clinical application, type II collagen hydrogels may be better because, as opposed to agarose, it degrades in time. Although culture in SF medium reduces the amount of proteoglycans produced during redifferentiation culture, isolating and expanding the cells in high osmolarity medium can largely compensate for this loss.