Fig 1. Overview of culturing protein-producing cells in three dimensional (3D) thermoreversible PNIPAAm-PEG hydrogels.

(A) The PNIPAAm-PEG polymers are soluble in water at low temperature (e.g. 4°C). They associate to form a network at high temperature (e.g. 37°C), resulting in a hydrogel. When the temperature is reduced (e.g. to 4°C), the polymers become soluble again and the hydrogel is liquefied. (B) To culture cells, single cells are mixed with 10% PNIPAAm-PEG solution at low temperature and casted onto a tissue culture plate, and then incubated at room temperature or 37°C for 5 minutes to form an elastic hydrogel before adding the warm medium. The single cells clonally expand into uniform spheroids within days. Proteins secreted from the cells can diffuse through the hydrogel into the medium. The conditioned medium is collected for purifying the proteins.