Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.
Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.
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DOI：
10.1093/humrep/12.5.1032
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发表时间：
1997-05
影响因子：
6.1
通讯作者：
O. Hovatta;R. Silye;R. Abir;T. Krausz;R. Winston
中科院分区：
文献类型：
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作者：
O. Hovatta;R. Silye;R. Abir;T. Krausz;R. Winston
从20名年龄在25-42岁的妇女中通过活检或卵巢切除术获得卵巢皮质组织。将其置于新鲜或冷冻保存后解冻的器官培养物中1-4个月。将组织切成直径为0.1-0.3 mm的切片，并转移到24孔培养板中的12 mm插入物中。将这些切片在α最低必需培养基（α-MEM）或具有添加的丙酮酸盐的Earle平衡盐溶液中培养4-21天。两种培养基均补充有10%人血清、胰岛素、促性腺激素和抗生素。一半的插入物用细胞外基质（Matrigel）预涂覆。组织学检查显示，所有培养物中均存在存活的、非闭锁的原始卵泡、初级卵泡和次级卵泡。次级卵泡颗粒细胞内可见有丝分裂。虽然闭锁卵泡的比例在培养过程中增加，但21天后仍存在非闭锁卵泡。4-11天后，在添加丙酮酸的Earle液中培养的活卵泡比例显著高于在MEM中培养的活卵泡比例（77%对38%，P < 0.001）。在有细胞外基质的培养物中，10-15天后活卵泡的比例显著高于无基质的培养物（85%对19%，P < 0.001）。冷冻卵巢组织解冻后培养不影响活卵泡的密度或比例。冷冻保存组织中三分之二的卵泡在培养10-15天后存活。结果表明，体外培养人初级卵泡和原始卵泡是可行的，冷冻保存的卵泡是有活力的。
Ovarian cortical tissue was obtained during gynaecological operations by biopsy or after oophorectomy from 20 women aged 25-42 years. It was placed in organ culture, either fresh or following thawing after cryopreservation, for 1-4 months. The tissue was cut in slices 0.1-0.3 mm in diameter and transferred to 12 mm inserts in 24-well culture plates. These slices were cultured for 4-21 days in either alpha minimum essential medium (alpha-MEM) or Earle's balanced salt solution with added pyruvate. Both media were supplemented with 10% human serum, insulin, gonadotrophins and antibiotics. Half of the inserts were precoated with extracellular matrix (Matrigel). Histological samples revealed that there were viable, non-atretic, primordial, primary and secondary follicles in all the cultures. Mitoses were seen in the granulosa cells of the secondary follicles. Although the proportion of atretic follicles increased during culture, non-atretic follicles were still present after 21 days. After 4-11 days the proportion of viable follicles was significantly higher when cultured in Earle's solution supplemented with pyruvate, than when cultured in MEM (77 versus 38%, P < 0.001). In cultures with extracellular matrix the proportion of viable follicles was significantly higher after 10-15 days than it was without matrix (85 versus 19%, P < 0.001). Culture after thawing frozen ovarian tissue did not affect the density or the proportion of the viable follicles. Two-thirds of follicles in cryopreserved tissue were viable after 10-15 days in culture. The results indicate that it is possible to culture human primary and primordial follicles in vitro, and follicles in cryopreserved tissue are viable.