Stimuli-responsive microwells for formation and retrieval of cell aggregates

Abstract

Generating cell aggregates is beneficial for various applications ranging from biotechnology to regenerative therapies. Previously, poly(ethylene glycol) (PEG) microwells have been demonstrated as a potentially useful method for generating controlled-size cell aggregates. In addition to controlling cell aggregate size and homogeneity, the ability to confine cell aggregates on glass adhesive substrates and subsequently retrieve aggregates from microwells for further experimentation and analysis could be beneficial for various applications. However, it is often difficult to retrieve cell aggregates from these microwells without the use of digestive enzymes. This study describes the stable formation of cell aggregates in responsive microwells with adhesive substrates and their further retrieval in a temperature dependent manner by exploiting the stimuli responsiveness of these microwells. The responsive polymer structure of the arrays can be used to thermally regulate the microwell diameters causing a mechanical force on the aggregates, subsequently facilitating the retrieval of cell aggregates from the microwells with high efficiency compared to PEG arrays. This approach can be potentially integrated into high-throughput systems and may become a versatile tool for various applications that require aggregate formation and experimentation, such as tissue engineering, drug discovery, and stem cell biology.

Fluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates

“…Given the important biophysical functionality, in vitro model systems would greatly benefit from in situ read-outs on local oxygen tension. These systems could for example reveal nutrient delivery problems during aggregate production in the context of tissue-engineering approaches [36,37] or help identifying interesting biological activity of drugs investigated in cell array cultures. [38] Fluorescent microbeads stably incorporated into a cell aggregate have great potential to provide these crucial data by making ratiometric measurements of an oxygen-sensitive luminophore with an oxygen-insensitive reference fluorophore.…”

Cell Detachment for Engineering Three-Dimensional Tissues

“…Various spheroid fabrication techniques that employ nonadherent surfaces [19], spinner flasks [20], hanging drop methods [21], micropatterning of cell-adhesive/non-adhesive surfaces [22,23], and micromolding [24,25] have been reported. Microwells consisting of thermoresponsive hydrogel have been used not only to generate spheroids but also to subsequently release them via a temperature-induced change in the hydrogel [26]. Spheroids have been harvested also with an electrochemical approach [13].…”

Intelligent recognitive systems in nanomedicine

“…In one application, the molds allow cell aggregates to be retrieved with higher efficiency than PEG molds by decreasing the temperature from 37°C to 24°C with the resulting swelling effectively pushing the aggregates out of the well [45]. The molds’ shape change at two temperatures also permits a sequential molding step allowing new spatial orientations like cylinders and cubes that contain two compartments.…”