Three-Dimensional Biofabrication Models of Endometriosis and the Endometriotic Microenvironment

Abstract

Endometriosis occurs when endometrial-like tissue grows outside the uterine cavity, leading to pelvic pain, infertility, and increased risk of ovarian cancer. The present study describes the optimization and characterization of cellular spheroids as building blocks for Kenzan scaffold-free method biofabrication and proof-of-concept models of endometriosis and the endometriotic microenvironment. The spheroid building blocks must be of a specific diameter (~500 μm), compact, round, and smooth to withstand Kenzan biofabrication. Under optimized spheroid conditions for biofabrication, the endometriotic epithelial-like cell line, 12Z, expressed high levels of estrogen-related genes and secreted high amounts of endometriotic inflammatory factors that were independent of TNFα stimulation. Heterotypic spheroids, composed of 12Z and T-HESC, an immortalized endometrial stromal cell line, self-assembled into a biologically relevant pattern, consisting of epithelial cells on the outside of the spheroids and stromal cells in the core. 12Z spheroids were biofabricated into large three-dimensional constructs alone, with HEYA8 spheroids, or as heterotypic spheroids with T-HESC. These three-dimensional biofabricated constructs containing multiple monotypic or heterotypic spheroids represent the first scaffold-free biofabricated in vitro models of endometriosis and the endometriotic microenvironment. These efficient and innovative models will allow us to study the complex interactions of multiple cell types within a biologically relevant microenvironment.

Keywords: 12Z; Kenzan; biofabrication; endometriosis; scaffold-free; spheroid.

Figure 1

Kenzan method of biofabrication. (A) A kenzan (Ansel Adams Museum, Yosemite National Park, CA, USA) used for flower arranging. Needles (white arrow) would hold the floral stems in place. (B) A Kenzan used for 3D biofabrication with the Regenova Bio 3D Printer. This Kenzan is made of a 9 × 9 stainless steel needle array (black arrow) and supported by two platens (arrowhead) to hold the needles in alignment and aid in removing the construct. (C) Workflow of Kenzan method biofabrication. Step 1—Generation of building blocks for biofabrication, called spheroids, large (~500 μm), tight, dense masses of cells that do not break up with gentle disruption. Step 2—Spheroids are placed onto the needles of the Kenzan with a robotic arm’s assistance. The X, Y, and Z placement on Kenzan is user-defined, allowing for a wide range of construct designs. Step 3—Spheroids are incubated in media on the Kenzan, allowing for a naturally secreted extracellular matrix to fuse adjacent spheroids. Step 4—The Kenzan is gently removed with one of the two platens’ aid, leaving behind a scaffold-free construct.

Figure 2

Large 12Z spheroids. (A) At 24 h, 18,000 12Z cells grown in ultra-low attachment round-bottom 96-well plates appear as a mass of cells with rough irregular edges (black arrowhead). (B) At 48 h, 12Z cells have formed a tight spheroid with a dense core (white arrow) and smooth rounded edges (white arrowhead). 12Z spheroids remain alive for at least 120 h, where the spheroids remained (C) proliferative (Ki-67, red) and (D) non-apoptotic (cCAS3, red). Blue = DAPI. Scale = 400 µm.

Figure 3

Large 12Z spheroids used for 3D biofabrication. Optimization for time post-seeding and the number of cells seeded for (A) roundness, (B) smoothness, and (C) diameter. (D) Representative image of a spheroid which fits all of the Regenova goal parameters. Scale = 400 µm. Grey shading indicates the tolerability range. N = 3.

Figure 4

12Z cells secrete increased inflammatory factors when grown as large spheroids but lose further sensitivity to TNFα. (A) Secreted inflammatory factors are increased in 12Z cells grown as large spheroids compared to monolayer. (B) Monolayer cells treated with TNFα secrete increased levels of inflammatory cytokines in a dose-response manner. (C) 12Z spheroids do not similarly respond, with only MCP1 secretion increasing after high doses of TNFα. Note the scale differs between graphs. N ≥ 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Estrogen-related gene expression is dependent on dimensionality in 12Z cells. CYP19A1 and HSD17β1, genes involved in estrogen synthesis, are upregulated in 12Z cells grown as spheroids compared to monolayer. ESR1, an estrogen receptor, is also increased in spheroids compared to monolayer. RQ = relative quantification. N ≥ 3. *p < 0.05, ** p < 0.01.

Figure 6

3D biofabrication of 12Z and HEYA8 spheroids into constructs. (A) 12Z spheroids immediately after biofabrication in a 3 × 3 × 2 pattern on the Kenzan. White dotted circle = individual spheroid. (B) Rendering and representative example of the 3 × 3 × 2 3D biofabricated cube made from 12Z spheroids < 24 h after removal from Kenzan. Scale = 200 µm. Arrow = hole left by Kenzan needle. (C) Rendering and representative example of a 3D printed tube made from 3 × 3 × 3 12Z spheroids > 24 h after removal from Kenzan. Scale = 200 µm. Arrowhead = hollow channel. (D) Rendering and representative construct from a 2 × 2 × 2 HEYA8 core surrounded by a 4 × 4 × 4 12Z shell. Scale = 200 µm.

Figure 7

Biofabrication of heterotypic spheroids. (A) Optimization of diameter for the total number of seeded cells and ratio of T-HESC to 12Z cells at 72 h post-seeding. The line indicates the tolerability range of Regenova technology. N = 3. (B) Spheroids made from 12Z and T-HESC cells self-assembled with 12Z-epithelial cells (Red) on the outside and T-HESC-stromal cells (Green) on the inside. Scale = 400 µm. 3 × 3 × 3 constructs biofabricated from heterotypic spheroids share similar self-assembly with (C) KRT-7 as an epithelial marker and (D) CD-10 as a stromal marker. Arrow = epithelial cells (12Z), arrowhead = stromal cells (T-HESC), dashed circle = presumed location of an individual spheroid. Scale = 50 µm. Inset Scale = 100 µm.