**1. Introduction**

In recent years, the use of tissue engineering technology for in vitro three-dimensional (3D) cell culture to construct 3D cell models for tumor research, drug screening, and tissue repair has aroused great interest in biomedical researchers and clinical workers [1–3]. Among them, scaffold materials play a key role. As the carrier of seed cells and bioactive growth factors, scaffold materials can provide a favorable microenvironment for cell growth, reproduction, metabolism, and other physiological activities, and induce cells to form a 3D model [4–7]. Therefore, an excellent scaffold material should have a 3D porous network structure to facilitate cell insertion and transport of nutrients and metabolic waste. At the same time, it should have ideal biocompatibility, degradability and mechanical strength matching the properties of the extracellular matrix.

Hydrogel is a kind of soft material with a 3D network structure, formed through various physical or chemical crosslinking methods [8–11]. Hydrogels have extremely similar physical properties to extracellular matrices, which have been widely used as scaffold materials for 3D cell culture [12–14]. Among them, thermosensitive polymersbased hydrogel scaffolds have attracted particular attention [15–17]. Thermosensitive

**Citation:** Liu, Y.; Luo, Y.-N.; Zhang, P.; Yang, W.-F.; Zhang, C.-Y.; Yin, Y.-L. The Preparation of Novel P(OEGMA-co-MEO2MA) Microgels-Based Thermosensitive Hydrogel and Its Application in Three-Dimensional Cell Scaffold. *Gels* **2022**, *8*, 313. https://doi.org/ 10.3390/gels8050313

Academic Editor: Esmaiel Jabbari

Received: 8 March 2022 Accepted: 17 May 2022 Published: 19 May 2022

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polymers generally appear liquid at room temperature, and can be evenly mixed with cells, drugs, growth factors, etc. When the temperature rises to its low critical dissolution temperature (LCST), or volume phase transition temperature (VPTT), and appropriate ions are present, the thermosensitive polymer undergoes in situ gelation to form injectable 3D hydrogels. Thus, it can nondestructively embed cells in situ and promote further cell growth. By cooling it to liquefy it, cells, or complex cell aggregates, can also be easily released.

Poly(N-isopropylacrylamide) (PNIPAM) is the most studied thermosensitive material with LCST or VPTT of 32 ◦C, and is often used as a scaffold material [18–20]. For instance, Ekerdt and colleagues successfully constructed a type of thermosensitive hyaluronic acid (HA)-PNIPAM brush polymer through thiol-ene click chemistry, which could mix with human embryonic stem cells (hESCs), or human induced pluripotent stem cells (hiPSCs), at 4 ◦C, and gel rapidly to form a hydrogel in situ at 37 ◦C [21]. After five days of culture, cells were able to grow rapidly and aggregate to form regular multicellular spheroids, while maintaining cell pluripotency after multiple passages. Moreover, this type of hydrogel was able to release the cells after cooling, liquefaction and centrifugation [22]. Liang et al. also synthesized a type of dendritic thermosensitive polymer from PNIPAM, dimeric glycerol, and polyethylene glycol (PEG). This polymer could also be used as a scaffolding material for the culture of hiPSCs, which were similarly able to release cells after simple cooling, liquefaction and centrifugation [23].

To further adjust and improve the properties of formed thermosensitive hydrogel, spherical thermosensitive polymers, namely microgels, were also used for the fabrication of hydrogel scaffolds. Microgels are spherical hydrogel nanoparticles with particle sizes from 10 nm to a few microns, which have the advantages of simple preparation, fast response and easy modification and are of considerable interest in the biomedical field [24–27]. Gan et al. successfully constructed this type of thermosensitive hydrogel scaffold using PNIPAM-based thermosensitive microgels for the first time. The hydrogel thus formed has an interconnected porous structure, which is highly conducive to the transport of oxygen, nutrients and cellular metabolites. Human embryonic kidney (HEK) 293T cells could be encapsulated in this hydrogel and grew well [28]. We also used PNIPAM-based microgels as scaffold materials to encapsulate aggregates of Human hepatocellular carcinomas HepG2 cells, in which multicellular spheroids of HepG2 cells could be quickly obtained [29]. In addition, Shen et al. also used the more hydrophilic NIPAM and acrylamide (AAm) copolymer microgels as scaffold materials for the culture of mouse melanoma cells [30]. It was found that the cells gradually grew in a single dispersed state. The larger the particle size of the microgels used, the better the activity of the cells. The Dai group also used PNIPAM-based microgels with a small negative charge and a small positive charge as cell scaffolds to study the culture and behavior of mouse embryonic mesenchymal stem cells (MSCS), respectively [31,32].

Despite the numerous advantages of PNIPAM thermosensitive polymers, the thermosensitive hydrogels formed from them suffer from a certain degree of shrinkage over time, which severely limits cell growth and activity. Although it is possible to reduce the shrinkage of the hydrogels by introducing acrylic acid (AA) with negative charge [33], blending PEG [29,34] and adjusting the particle size of the microgels [35], the effect is still not satisfactory. In addition, PNIPAM polymers are toxic to some extent and difficult to degrade. Therefore, it is an urgent problem to find a type of thermosensitive gel material with low shrinkage, and excellent biocompatibility and biodegradability. In recent years, novel thermosensitive polymers based on oligo(ethylene glycol) methacrylate (OEGMA) and 2-(2-methoxyethoxy) ethyl acrylate (MEO2MA) have aroused great interest from researchers because of their excellent biocompatibility and biodegradability [36,37]. OEGMA and MEO2MA are two types of PEG-based macromolecular monomers with similar structures, in which the LCST of POEGMA homopolymer is about 90 ◦C and often acts as a hydrophilic chain segment, while PMEO2MA homopolymer has an LCST of about 19 ◦C and often acts as a hydrophobic chain segment. The copolymers formed from them also

have outstanding thermosensitive behavior. The LCST or VPTT could vary from 19 to 90 ◦C by simply adjusting the relative proportions of OEGMA and MEO2MA in the polymerization process. Several studies have reported using these novel thermosensitive polymers for the culture of various types of cells. For instance, Anderson et al. prepared a brush-like P(OEGMA-co-MEO2MA) thermosensitive polymer by adjusting the ratio of OEGMA and MEO2MA. It was used as a matrix material to study the effect on adhesion and morphology of L-929 mouse fibroblasts [38,39]. It was found that the combination of thermosensitive polymers with a smaller relative proportion of hydrophilic OEGMA was more favorable for cell growth and adhesion. In addition, Bakaic et al. [40] constructed an injectable hydrogel scaffold with temperature and pH dual responsiveness using chemical crosslinking between neutral or charged aldehyde functionalized POEGMA copolymers and hydrazide functionalized POEGMA copolymer precursors. They found that the charged hydrogel was more supportive of the two-dimensional (2D) cell adhesive of 3T3 mouse fibroblasts and the 3D stabilization and proliferative of ARPE-19 human retinal epithelial cells.

In this manuscript, we discuss the feasibility of using P(OEGMA-co-MEO2MA) spherical microgels to construct thermosensitive hydrogel 3D cell scaffolds for the first time. We first used OEGMA and MEO2MA to prepare a series of P(OEGMA-co-MEO2MA) microgels with different VPTT and sizes through free radical polymerization. Then, novel P(OEGMA-co-MEO2MA) microgels-based thermosensitive hydrogels were prepared by the thermal induction method under conditions of heating and the presence of salts. MCF-7 human breast cancer cells were used as the model cells for in-situ embedding and 3D co-culture in this novel thermosensitive hydrogel. To the best of our knowledge, there are no reports related to P(OEGMA-co-MEO2MA)-based microgels as scaffolds for 3D cell culture. Therefore, our study may provide new ideas for the development of thermosensitive hydrogel scaffolds.

### **2. Results and Discussion**
