Three-Dimensional Magnetic Bioprinting Spheroids as an In Vitro Model to Study the Oviductal Physiology

Abstract

In vitro models to study the oviduct are challenged by cellular dedifferentiation, a complex coculture system for embryo production, limited cell lifespan, and/or very complex methodologies. Hence, we aimed to develop an in vitro oviductal model using the magnetic bioprinting system, a three-dimensional (3D) culture system. Using the bovine epithelial and stromal oviductal cells (BOEC and BOSC, respectively), we produced the Oviductal Magnetic Spheroid (OMS), a duo somatic cell spheroid aggregate with self-organization capacity. The OMS showed to be viable for 21 days and recapitulated the oviductal tissue features after 7 days in culture, such as a simple epithelial cell layer facing outwards, expressing ciliation (acetylated tubulin positive) and secretory marker (oviduct-specific glycoprotein 1). Although the responsiveness for hormonal treatment with estradiol and progesterone in an estrous cycle-dependent way might require further improvements, the OMS offers an ethical and practical alternative as a three-dimensional oviductal in vitro model to study oviductal physiology, and maybe, a future platform to test therapies and a technology aiming to improve fertility and assisted reproduction success.

Keywords: OVGP1; bioprinting; cytokeratin; extracellular matrix; vimentin.

Figure 1

Elementary standardization steps for spheroid formation: spheroid cell number size and cellular magnetization methods. (A) Schematic representation of magnetic 3D Bioprinting (m3DB) system; (B) Spheroid cell number size evaluation (Experiment I) at 48 h after cell seeding: (i) 50,000, 25,000, 10,000, and 5000 cells per well formed by BOEC‐only (100% BOEC, white bars) or BOEC/BOSC‐mix (70/30%, blue bars) stained with Hoechst 33342 (nuclei) and Propidium Iodate (PI, necrotic‐positive cell marker); (ii) schematic representation of the necrotic zone formation in big spheroids ( > 25,000 cells); (iii) schematic representation of absence of necrotic zone formation in small spheroids ( < 10,000 cells); (iv) 5000 cells spheroids (representative images, bright‐field), and the graphical results of spheroid area (square millimeter—mm²) and spheroid compaction level (percentage, %), (v) 10,000 cells spheroids (representative images, bright‐field), and the graphical results of spheroid area (mm²) and spheroid compaction level (%), ** and *** indicates p < 0.01 and 0.001, respectively; (C) Cellular magnetization methods evaluation (Experiment II) at 48 h after cell seeding: (i) schematic representation of cell magnetization by overnight incubation (ON) or centrifugation (C), starting with the cell in monolayer culture, followed by trypsinization step (T) before being seeded in the 96‐well non‐adherent plate and incubated with the Magnetic Bioprinting Plate; (ii) 10,000‐cell spheroids: representative images (bright‐field) of BOEC‐only (100%BOEC) or BOEC/BOSC‐mix (70/30%) and the graphical results of spheroid area (mm²) and spheroid compaction level (%), different letters represent statistical differences (p < 0.05) among magnetization methods in BOEC‐only (a‐b) or BOEC/BOSC‐mix (x‐y), **** represents statistical differences (p < 0.0001) when comparing BOEC‐only vs. BOEC/BOSC‐mix spheroids under the same magnetization methods. All data are presented as mean ± standard deviation (SD) with single spheroid values represented in the graphs as a circle. All scale bars represent 100 μm. Abbreviations: BOEC, bovine oviduct epithelial cells; BOSC, bovine oviduct stromal cells.

Figure 2

Improvement of spheroid formation: cell seeding protocols and cell proportion (BOEC/BOSC). (A) Schematic representation of one‐ or two‐step cell seeding protocols, showing the cell seeding order and the duration of each step, (B) results of experiment III of 10,000‐cell spheroids evaluated at 24, 48, and 144 h after cell seeding (i) representative images (bright‐field) of the spheroids formed with BOEC‐only (100%BOEC, white bars) and BOEC/BOSC‐mix (70/30%, lighter blue bars, and 50/50%, darker blue bars) at three time‐points and the graphical results of (ii) spheroid area (mm²), (iii) spheroid compaction level (%), and (iv) immunostaining in 144 h spheroids for epithelial cell marker (anti‐cytokeratin, green color) and Hoechst 33342 (DNA, blue); different letters represent statistical differences (p < 0.05) among different cell proportion in one‐step (a‐c) or two‐step (x‐y) protocols, * (p < 0.05) and **** (p < 0.0001) represents statistical differences when comparing cell seeding protocols (one‐ vs. two‐step). All data are presented as mean ± standard deviation (SD) with single spheroid values represented in the graphs as a circle. All scale bars represent 100 μm. Abbreviations: BOEC, bovine oviduct epithelial cells; BOSC, bovine oviduct stromal cells.

Figure 3

Spheroid stability with culture media supplementation with extracellular matrix (ECM). (A) schematic representation of the experimental design, 10,000‐cell spheroids consisting of BOEC/BOSC‐mix (70/30%) were seeded as one‐ or two‐step protocol with culture media supplemented with 0, 0.5, 1, or 2% v/v Geltrex supplementation, cells were incubated in the supplemented media and on top of the Magnetic Bioprinting Plate for 48 h, following the culture without them until 216 h in total. (B) Analysis at 48 h of culture: (i) representative images (bright‐field), (ii) representative images of spheroids stained with Hoechst 33342 (nuclei) and Propidium Iodate (PI, necrotic‐positive cell marker), graphical results of (iii) spheroids area (square millimeter, mm²), (iv) spheroid compaction level (percentage, %), and (v) necrotic‐positive cell ratio (%); different letters represent statistical differences (p < 0.05) among different ECM supplementation groups in one‐step (a‐c) and two‐step (x‐y) protocols and * p < 0.05, ** p < 0.01, and **** p < 0.0001 represent statistical differences when comparing one‐ vs. two‐step protocols. (C) Analysis of spheroids cultured with 1% v/v Geltrex supplementation at 48 and 216 h of culture: (i) representative images of spheroids (bright‐field and Hoechst + PI), graphical results of (ii) spheroids area (mm²), (iii) spheroid compaction level (%), and (iv) necrotic‐positive cell ratio (%); different letters represent statistical differences (p < 0.05) among different culture time in one‐step (a‐b) and two‐step (x‐y) protocols and *** represents statistical difference (p < 0.001) when comparing one‐ vs. two‐step protocols. All data are presented as mean ± standard deviation (SD) with single spheroid values represented in the graphs as a circle. All scale bars represent 100 μm. Abbreviations: BOEC, bovine oviduct epithelial cells; BOSC, bovine oviduct stromal cells; NS, NanoShuttle‐PL.

Figure 4

Long‐term spheroid viability evaluation: 10,000‐cell spheroids consisting of BOEC/BOSC‐mix (70/30%) were seeded as a two‐step protocol with culture media supplemented with 1% v/v Geltrex supplementation, cells were incubated in the supplemented media and on top of the Magnetic Bioprinting Plate for 72 h (considered day zero) and followed until Day 28 of culture. (i) representative images of spheroids (bright‐field and Hoechst + PI) on Days 1, 7, 14, 21, and 28 of culture, graphical results of (ii) spheroid area (mm²), (iii) spheroid compaction level (%), and (iv) necrotic‐positive cell ratio (%); different letters (a‐b) represent statistical differences (p < 0.05) among different culture days. All data are presented as mean ± standard deviation (SD) with single spheroid values represented in the graphs as a circle. All scale bars represent 100 μm. Abbreviations: BOEC, bovine oviduct epithelial cells; BOSC, bovine oviduct stromal cells; NS, NanoShuttle‐PL.

Figure 5

Characterization of the oviductal magnetic spheroid (OMS) structure organization. (A) Spheroids of 10,000‐cell size consisting of BOEC/BOSC‐mix (70/30%) were seeded as one‐ or two‐step protocols with culture media supplemented with 1% v/v Geltrex supplementation, cells were incubated in the supplemented media, and on top of the Magnetic Bioprinting Plate for 72 h, at this time the magnetic plate was removed and the culture media was changed (defined as day zero). Representative images of OMS at Day 1 and Day 7 from one‐ and two‐step protocols stained for anti‐cytokeratin (green, epithelial cell marker), anti‐vimentin (red, stromal cell marker), Hoechst 33342 (blue, DNA), and merged images (Maximum Intensity Z‐projection). The right panel represents the native oviductal tissue (cryosection of the ampulla segment) stained with the same markers. (B) Representative images from Supporting Information S6: Movie 1, Maximum Intensity Z‐projection (left panel) and selected z‐stacks (right panel) stained with anti‐cytokeratin, anti‐vimentin, and Hoechst, OMS at Day 7 from the two‐step protocol. (C) Spheroids of 10,000‐cell size consisting of BOEC/BOSC‐mix (70/30%) seeded as a two‐step protocol with culture media supplemented with 1% v/v Geltrex supplementation, cells were incubated in the supplemented media, and on top of the Magnetic Bioprinting Plate for 72 h, at this time the magnetic plate was removed, and the culture media was changed (defined as day zero). Representative images of paraffin sections of OMS at Day 7 (top panel) and Day 14 (middle panel), and a native oviductal tissue (bottom panel, ampulla segment). Samples were stained with Hoechst 33342 (DNA in blue, left panel), toluidine blue at pH 2.5 (middle panel), or toluidine blue at pH 4 (right panel). Scale bars are indicated in each picture; otherwise, white scale bars represent 100 μm, and black scale bars represent 20 μm. The symbols in the images indicate the oviductal lumen (*), stromal cell layer (arrowhead), epithelial cell layer (arrow), and muscular layer (#).

Figure 6

Characterization of cell ciliation and secretion markers in OMS formed by cells harvested at different estrous cycle phases. OMS formed by BOEC and BOSC harvested from oviductal tissues at follicular or luteal phases of the estrous cycle and cultured for 14 or 21 days (i) representation of the experimental design of OMS formation and culture, (ii) representative images of OMS stained with anti‐cytokeratin (green, epithelial cell marker) and Hoechst 33342 (blue, DNA) and merged images, (iii) representative images of OMS in bright‐field and co‐stained with Hoechst 33342 (nuclei) and Propidium Iodate (PI, necrotic‐positive cell marker), graphical results of (iv) spheroids area (square millimeter, mm²), (v) spheroid compaction level (percentage, %), and (vi) necrotic‐positive cell ratio (%), (vii) representative images of OMS stained with anti‐acetylated alpha‐tubulin (acTUB, green, cilia marker), anti‐oviduct‐specific glycoprotein 1 (OVGP1, red, secretion marker), and Hoechst 33342 (blue, DNA) and merged images, low and high magnifications (inserts) and graphical results of relative fluorescent intensity of (viii) acTUB and (ix) OVGP1 presented as arbitrary unit (A.U.) of the specific pixel quantification per spheroid area; different letters represent statistical differences (p < 0.05) among different days of culture in OMS from follicular (a‐b) and luteal (x‐y) phases and **** represents statistical difference (p < 0.0001) when comparing follicular versus luteal phases. All data are presented as mean ± standard deviation (SD) with single spheroid values represented in the graphs as a circle. Scale bars represent 100 μm (white bars) or 50 μm (gray bars). Abbreviations: BOEC, bovine oviduct epithelial cells; BOSC, bovine oviduct stromal cells.

Figure 7

Hormone responsiveness of OMS. 10,000‐cell spheroids of BOEC/BOSC‐mix (70%/30%) were formed using the two‐step protocol. OMS was formed with cells harvested during the luteal phase of the estrous cycle and evaluated during different days of culture or submitted to hormonal treatment. (A) OMS from different culture days, not treated, and (B) OMS submitted to hormone treatment: progesterone (P4, 100 ng/mL) for seven days to mimic a luteal phase, followed by estradiol (E2, 300 pg/mL) for three days to mimic a follicular phase, followed by four days with no hormone in the media, simulating the post‐ovulatory period. (i) Schematic representation of the experimental designs; (ii) AcTUB and OVGP1 protein quantification (fluorescence intensity per area, presented as an arbitrary unit, A.U.); * and ** represent statistical difference (p < 0.05 and < 0.01, respectively). (iii) Relative mRNA abundance of the genes forkhead box protein J1 (FOXJ1, ciliation marker), oviduct‐specific glycoprotein 1 (OVGP1, secretion marker), estrogen receptor 1 and 2 (ESR1 and ESR2, respectively), progesterone receptor (PGR), E2F Transcription Factor 1 (E2F1), and Epidermal Growth Factor Receptor (EGFR), presented as an arbitrary unit (A.U.); *, **, and *** represent statistical difference (p < 0.05, < 0.01, and < 0.001, respectively). All data are presented as mean ± standard error (SE) with single values represented by the black circles.