환경제어가 가능한 인베스 3D 세포 프린팅 기술을 통한 지방세포로 조밀하게 채워진 지방 구조체의 개발

Abstract

Adipose tissue is a representative loose connective tissue composed mostly of lipid-accumulating adipocytes, which differentiate from preadipocytes. Adipose tissue is densely packed with mature adipocytes, and this morphology is associated with its functionality. Conventionally, adipose tissue plays a role in fat deposition, protection of neighboring organs, and insulation. However, adipose tissue has recently been identified as a major endocrine organ. The adipose tissue coordinates with several organs through numerous adipokines secreted from mature adipocytes and is responsible for overall health. Consequently, morphological and functional recapitulation of adipose constructs is becoming gradually important in biological and pathological studies. Although direct use of adipocytes results in immediate recapitulation of native functions, their fragile properties result in many dead cells during fabrication. Moreover, it remains challenging limitations to recapitulate the morphology of native adipose tissue with fully populated adipogenic lipid droplets, mainly due to poor adipogenesis resulted from the low cell amount in a unit area of current adipose constructs. This thesis suggests the use of environmentally controlled in-bath three-dimensional (3D) cell printing to engineer morphologically and functionally biomimetic densely packed adipose tissues in vitro. To achieve this, a hybrid bioink containing alginate was first developed for bath suspension, because alginate is a well-known biomaterial that lacks cell-binding motifs. It was hypothesized that alginate in the hybrid bioink would provide in-bath printed cells with a cell-unfriendly environment, which regulates cell migratory behavior to allow dominant proliferation in the printed region. In addition, we hypothesized that selectively proliferated preadipocytes would form a densely packed adipose tissue construct after preadipocyte differentiation. The optimal concentration of the hybrid bath bioink (1% alginate + 1.5% adipose-derived decellularized matrix) was determined by rheological assessments, shape maintenance ability, and selective cell proliferation capability. In the hybrid bath bioink, preadipocytes (printed at >107 cells/ml) proliferated without cell migration towards the bath, forming a densely populated cellular constucts. After adipogenesis, the morphological and quantitative results demonstrated that the selectively proliferated preadipocytes could differentiate into mature adipocytes. The resulting in vivo-like densely packed adipose tissue was successfully engineered in vitro when compared with conventional approaches. The levels of representative hormones secreted by the mature adipocytes were measured. The results showed that the densely packed adipose tissue construct secreted the highest levels of adipokines among the experimental groups, indicating that organotypic cell-to-cell interactions enhance adipose tissue maturation. The densely packed adipose tissue construct also showed pathological changes associated with obesity under relevant conditions found in obese patients. The adipose tissue shows obesity characteristics under a prolonged hyperglycemic environment, leading to an increase in adipocyte size (adipocyte hypertrophy). We applied the obesity-inducing condition to the densely packed adipose tissue construct to determine if the phenomenon could be embodied. Apparent adipocyte hypertrophy was observed in our construct compared with the healthy adipose tissue construct cultured under normal glucose conditions. Most obesity-associated complications result from insulin resistance in hypertrophic adipocytes. The staining results showed few activated insulin receptors in the obese adipose tissue construct. Glucose uptake tests showed no notable changes upon insulin treatment of the obese adipose tissue construct. These outcomes revealed that the hallmarks of obese adipose tissue were successfully induced in the adipose tissue construct under obesity-inducing conditions. Furthermore, we recapitulated obesity-induced adipose tissue inflammation in vitro through coculture with monocytes. The interaction between hypertrophic adipocytes and monocytes significantly increased pro-inflammatory cytokine secretion in the obese adipose tissue construct with monocytes. We expected that this pro-inflammatory response would induce monocytes to differentiate into M1 macrophages as an adapted phenotype to the physiological state. To confirm this, an immunostaining was performed using CD206 and CD68 antibodies against M2 and M1 macrophages, respectively. The results demonstrated the predominant differentiation of monocytes to M1 macrophages in the obese adipose tissue construct. Interestingly, crown-like structures, the representative histological hallmark of obesity-associated inflamed adipose tissue, were discovered in the horizontal cross-sectional areas of the obese adipose tissue construct cocultured with monocytes. In summary, this thesis suggests an environmentally controlled in-bath three-dimensional (3D) cell-printing technique to create native-mimetic adipose tissues in vitro. To this end, a hybrid bath bioink developed for inducing selective cell proliferation was first developed. The proposed technique enabled preadipocytes to be located in a predesigned restricted region, and they became a densely packed adipose tissue construct consisting of native mimetic adipocytes. The achieved construct could recapitulate the pathological changes associated with obesity under prolonged nutrient availability. Monocytes cocultured with the obese adipose tissue construct differentiated into M1 macrophages, resulting in chronic inflammation in the adipose tissue. When cocultured with monocytes, they differentiated predominantly into M1 or M2 macrophages as per their resident states. The crown-like structure demonstrated the successful recapitulation of chronically inflamed adipose tissue in vitro. Overall, the proposed cell printing strategy shows potential as a reliable engineering platform not only for deepening the biological understanding of adipose tissue, but also for addressing several diseases related to adipose tissues, such as obesity.