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Hormone autocrination by vascularized hydrogel delivery of ovary spheroids to rescue ovarian dysfunctions

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Science Advances  28 Apr 2021:
Vol. 7, no. 18, eabe8873
DOI: 10.1126/sciadv.abe8873
  • Fig. 1 Ovary cells and artificial follicle spheroids from rats.

    (A) (Left) The schematic structure of the native ovarian follicle was mimicked by (right) two forms of artificial spheroids in which granulosa (G) cells were encapsulated by a theca (T) cell layer (T layer) without (top, GT) or with (bottom, GMT) Matrigel (M) as a basal lamina (BL) mimetic. G and T cells were isolated from ovarian tissues of female rats (3 weeks old) by Percoll gradient separation. Multilayer cell spheroids were produced by first forming G cell spheroids in AggreWell, followed by Matrigel (M) coating and/or T cell loading onto the spheroids. (B) Before spheroid formation, the phenotypic purity was enhanced by a series of subculturing and then determined by examining the protein expression of CYP19 (aromatase, G cell marker) and CYP17A (17, 20 lyase, T cell marker), respectively. The phase contrast and immunofluorescence images were obtained and quantitatively analyzed using ImageJ (AU, arbitrary unit) as an indication of successful purification of each cell type. Scale bar, 200 μm. (C) The results were confirmed by flow cytometry with quantitative analysis after staining of G and T cells with anti-CYP19–FITC (G cell) and anti-CYP17A1–PE (T cells) antibodies, respectively. (D) 3D structures of GMT and GT spheroids were visualized by confocal imaging after labeling G (red) and T (green) cells with CellTracker(s). Scale bar, 100 μm. (E) The numbers of G and T cells in GMT spheroids were the same as those of GT spheroids. Superior spheroid functions of GMT over GT were determined by examining the protein expression of cell proliferation markers (PCNA and cyclin D1) and the secretion of estrogen (FSH-R) and progesterone (LH-R) by Western blot with quantitative analysis. Both types of spheroids were cultured at AggreWell for 2 weeks without gelatin coating and perfusion. Data are presented as means ± SEM. **P < 0.01, ***P < 0.001 between lined groups.

  • Fig. 2 VHOS for spheroid culture in a 3D channel network hydrogel.

    (A) An implantable VHOS was produced by forming perfusable 3D channel networks inside for spheroid culture through the step-by-step procedures as shown in the scheme. (Left) (i) A PDMS chamber was casted by pouring into a 3D printed mold. (ii) Threads of thermo-responsive PNIPAM fibers were produced by spinning and placed into the PDMS chamber. (iii) Gelatin solution was mixed with G(M)T spheroids and poured to cover the fiber threads in the chamber, followed by enzyme-crosslinked gelation. (iv) The PNIPAM fibers were dissolved out at 37°C to form a perfusable channel network in the gelatin gel. (Right) Cell spheroids were cultured by perfusing culture medium into channel networks continuously at a flow rate of 20 μl/min using a peristaltic pump (dynamic) or through gravity-based medium flow (static). A 30-day culture was conducted to mimic a 30-day menstrual cycle. (B) Cell viability in spheroids was maintained in a microchannel hydrogel for 30-day perfusion culture as shown by confocal images (green, live cell; red, dead cell; blue, nucleus; purple, channel network) after live/dead staining together with perfusion staining of the channel network in the hydrogel (left). 3D structures of GMT spheroid were visualized by confocal imaging after labeling G (red) and T (green) cells with CellTracker(s) (right). Scale bar, 100 μm. (C) The 30-day culture’s effects on GMT and GT spheroids in dynamic versus static condition were determined by examining (left) structural stability and expansion of spheroids, (middle) cell viability and proliferation, and (right) hormone secretion through medium collection every other day. Data are expressed as means ± SEM. *P < 0.05, GT static versus GMT static; P < 0.05, GT dynamic versus GMT dynamic; #P < 0.05, GMT static versus GMT dynamic.

  • Fig. 3 Rats underwent both ovariectomy and hindlimb surgery at day 7, and hydrogel implantation at day 14 or hormone injection at day 28 (every 4- to 5-day repetition), followed by hormone level measurement every seventh day until euthanization (day 63).

    (A) Rats underwent either sham operation (group 1: Ovary-intact, n = 3) or ovariectomy. Ovariectomized rats were injected with E without (group 2: OVX + E, n = 5) or with P (group 3: OVX + E + P, n = 6). (B) Ischemia-mediated perfusion connection between the microchannels and host vessels enables GMT spheroids to release hormones into the blood circulation (group 4: OVX + VHOS, n = 5). Consequently, the endocrine feedback mechanism is activated to treat ovariectomy by VHOS implantation. The last group is OVX with no treatment (group 5: OVX, n = 5). (C) The blood perfusion ratio was recovered at day 14 after VHOS implantation, as determined by laser Doppler perfusion imaging (each group, n = 3). (D) Confocal imaging (scale bar, 50 μm) and (E) perfusion staining of microchannels showed maintenance of GMT viability for 28 days. (F) The endocrine function recovery was determined by the circulating plasma levels of hormones. Data are means ± SEM. *P < 0.05 versus group 1; P < 0.05 versus group 5.

  • Fig. 4 Endometrium regeneration by VHOS.

    VHOS implantation regenerated the endometrium to the levels of the Ovary-intact group as opposed to the OVX group (n = 3 to 6 rats). (A) Top: The morphological images (scale bar, 1 cm) were further examined by (middle) H&E staining (scale bar, 200 μm) with (bottom) quantitative analysis (Ovary-intact, n = 4; OVX + E, n = 3; OVX + E + P, n = 3; OVX + VHOS, n = 3; OVX, n = 3). (B) Top: Ultrasonography images of uterus (original, 100×) were also obtained with magnification and labeling (200×), and then (bottom) the endometrium area of each group was quantitatively analyzed. Endometrium thickness and uterus index were compared among the test groups (each test group, n = 3). Data are means ± SEM. *P < 0.05 versus Ovary-intact; P < 0.05 versus OVX. (Photo credit: Hyo-Jin Yoon, Yonsei University College of Medicine.)

  • Fig. 5 VHOS-mediated attenuation of side effects from hormone therapy.

    The advantage of VHOS implantation was shown by presenting the side effects (i.e., hyperplasia, cancerous progress, and deep vein thrombosis) of the hormone treatment groups (Ovary-intact, n = 4; OVX + E, n = 5; OVX + E + P, n = 6; OVX + VHOS, n = 5; OVX, n = 5), as supported by the following data. (A) H&E staining (scale bar, 100 μm) with quantitative analysis of hyperplasia area (left) and number (right). White arrows indicate endometrial hyperplasia, a potential inducer of uterine cancer. (B) Expression of p53 and PTEN as indications of endometrial carcinoma progress and suppression, respectively, by (top) IHC images (scale bar, 100 μm) with (middle) quantitative analysis (each test group, n = 4) and (bottom) Western blot with quantitative analysis. Data are means ± SEM. *P < 0.05 versus Ovary-intact; P < 0.05 versus OVX. (C) TEM analysis of deep vein thrombosis after ligation of inferior vena cava (scale bar, 5000 nm).

  • Fig. 6 Prevention of menopause-related health risks using VHOS implantation.

    (A) Gaining body weight and fat percentage, as indications of menopause risk, for 8 weeks (Ovary-intact, n = 4; OVX + E, n = 5; OVX + E + P, n = 6; OVX + VHOS, n = 3; OVX, n = 5). (B) Losses of BMD and BMC as other side effects of menopause, analyzed by quantitative analysis of DEXA scans (Ovary-intact, n = 4; OVX + E, n = 5; OVX + E + P, n = 6; OVX + VHOS, n = 3; OVX, n = 5). (C) Qualitative scan images (top) of the femoral micro-architecture by micro-CT with quantitative image analyses of four bone density indexes (BV/TV, bone volume/total volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation) from a 3D reconstruction area of rat femoral bone, comparing the density (red arrow) and cavity (white arrow) in trabecular bone fracture with those of OVX group (Ovary-intact, n = 4; OVX + E, n = 5; OVX + E + P, n = 6; OVX + VHOS, n = 5; OVX, n = 5). Data are means ± SEM. *P < 0.05 versus Ovary-intact; P < 0.05 versus OVX.

Supplementary Materials

  • Supplementary Materials

    Hormone autocrination by vascularized hydrogel delivery of ovary spheroids to rescue ovarian dysfunctions

    Hyo-Jin Yoon, Yong Jae Lee, Sewoom Baek, Young Shin Chung, Dae-Hyun Kim, Jae Hoon Lee, Yong Cheol Shin, Young Min Shin, Chungsoon Ryu, Hye-Seon Kim, So Hyun Ahn, Heeyon Kim, Young Bin Won, Inha Lee, Myung Jae Jeon, Si Hyun Cho, Byung Seok Lee, Hak-Joon Sung, Young Sik Choi

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