Templated Macroporous Polyethylene Glycol Hydrogels for Spheroid and Aggregate Cell Culture

Abstract

Macroporous cell-laden hydrogels have recently gained recognition for a wide range of biomedical and bioengineering applications. There are various approaches to create porosity in hydrogels, including lyophilization or foam formation. However, many do not allow a precise control over pore size or are not compatible with in situ cell encapsulation. Here, we developed novel templated macroporous hydrogels by encapsulating uniform degradable hydrogel microspheres produced via microfluidics into a hydrogel slab. The microspheres degraded completely leaving macropores behind. Microsphere degradation was dependent on the incubation medium, microsphere size, microsphere confinement in the hydrogel as well as cell encapsulation. Uniquely, the degradable microspheres were biocompatible and when laden with cells, the cells were deposited in the macropores upon microsphere degradation and formed multicellular aggregates. The hydrogel-encapsulated cell aggregates were used in a small drug screen to demonstrate the relevance of cell–matrix interactions for multicellular spheroid drug responsiveness. Hydrogel-grown spheroid cultures are increasingly important in applications such as in vitro tumor, hepatocellular, and neurosphere cultures and drug screening; hence, the templated cell aggregate-laden hydrogels described here would find utility in various applications.

Micro-Clotting of Platelet-Rich Plasma Upon Loading in Hydrogel Microspheres Leads to Prolonged Protein Release and Slower Microsphere Degradation

“…We have previously shown that by simply controlling disperse and continuous phase flow rates we can achieve microspheres in the range of~100-500 µm (as measured directly upon fabrication and before swelling) [41]. This is a useful range, since microspheres with diameters of 50-300 µm have shown suitable for localized drug delivery via injection [42,43].…”

Engineering Multi‐Cellular Spheroids for Tissue Engineering and Regenerative Medicine

“…[108] Another study used cells that internalized iron oxide nanoparticles to effectively fabricate human MSC spheroids, and it was confirmed that an in vitro stem cell niche has been successfully mimicked by collagen gels embedded with magnetized spheroids. [109] An alternative to the [108] Iron oxide Human MSC [ 20,109] Polymeric micro-/nanosphere PLGA Human MSC [19,21] Gelatin Rat MSC, human ASC, human MSC [22,120,121,122] PDMS Human MSC [23] Collagen Rat hepatocytes [123] Polyacrylamide Human ASC [124] PCL Human MSC, human dermal fibroblasts [125] Hydrogel Matrigel, collagen, gelatin HepG2 [127] PNIPAAm-PEG Human PSC [128] Chitosan-PEG-genipin Glioblastoma cells [129] PEG U87, primary HDF, PC-12, INS-1, U251, human MSC [ 24,25,130] Alginate HepG2, mouse ESC, human ASC, HeLa cell [ 131,132,134,135] Heparin Mouse ESC [133] Cell membrane modification PNIPAAm HL-60, rat heart myoblasts, HepG2 [ 17,114] Oxyamine, ketone Human neonatal dermal fibroblasts, NIH3T3, 3T3 swiss albino mouse fibroblasts, human MSC [115] DNA Human luminal cells, myoepithelial cells, epithelial cells, HUVEC, human MSC [18] Fibronectin, gelatin Pancreatic cell, human pancreatic cancer cell, HUVEC, MRC-5 [ 116,117] Hyaluronic acid Renal mesangial cells [118] Gelatin, alginate, chitosan Human breast cancer cells [119] Nanofiber PLGA Embryonic kidney cells, dermal fibroblasts [ 136,137] PLLA Dermal fibroblasts, human MSC…”

“…[ 24 ] U251 cells were also encapsulated in PEG hydrogel microspheres fabricated via microfluidic devices to produce regular‐size spheroids of around 100 µm. [ 25 ] In one study, a two‐layer microfluidic device with multiple channels was developed to fabricate human MSC‐loaded PEG microspheres, which increased the microsphere fabrication efficiency by more than 600% over conventional microfluidic devices. [ 130 ] Size‐controlled alginate hydrogel microspheres containing HepG2 cells were fabricated using a concave mold and spontaneous spheroid formation was observed inside the hydrogel microspheres.…”

“…Furthermore, hydrogels with biocompatible chemistry have been developed to produce microgels for the fabrication of size-controlled spheroids with high efficiency. [24,25] Thus, it is clear that advances in biomaterials have contributed to overcome the drawbacks of general spheroid culture methods and to increase the potential of spheroids as therapeutic agents in regenerative medicine.…”

Microfluidic technologies to engineer mesenchymal stem cell aggregates—applications and benefits

“…If the degradation rate of a hydrogel is equal to the regeneration of the damaged tissue, multicellular aggregates can pop out of the hydrogel and help the damaged tissue to be recovered. Combining micro and macromaterials stimulate microniche generation, enabling stem cell differentiation into a specific lineage (Kamperman et al, 2017a;Imaninezhad et al, 2018). A summary about the use of droplet-based microfluidic devices for the formation of different types of microgels is presented as Table 1.…”