目的:是否有可能使用冷冻保存的子宫内膜异位症组织碎片建立离体子宫内膜异位症模型?
结论:冷冻保存的子宫内膜异位症组织碎片在解冻后和培养至少3天期间仍然存活,因此可用于建立离体子宫内膜异位症模型以有效地测试潜在的治疗药物。
背景:子宫内膜异位症是最常见的良性妇科疾病,具有巨大的社会负担;然而,治疗仍然缺乏。为了有效测试潜在的新疗法,基于先前冷冻保存的子宫内膜异位症组织的离体模型非常理想,该模型可以概括不同的子宫内膜异位症亚型及其微环境。
方法:通过手术切除从28例患者中获得三种不同亚型的子宫内膜异位症组织碎片。冷冻保存和解冻后,评估这些组织碎片的活力和代谢活性。手术切除后直接将活力与11例患者的新鲜碎片进行比较。在来自两名患者的冷冻保存和解冻的组织碎片中进行实验干预研究,以确认这些组织用于离体干预研究的可用性。
方法:根据三种不同方案冷冻保存子宫内膜异位组织碎片(n=45)。解冻后,片段培养24小时。进行基于刃天青的测定以评估组织片段的代谢活性。此外,通过VivaFix分析细胞类型特异性活力,Hoechst33342和α-平滑肌肌动蛋白免疫荧光染色和共聚焦显微镜。根据苏木精-伊红染色在组织学上证实了子宫内膜异位症的存在。用吡非尼酮或二甲双胍处理冷冻保存和解冻的组织片段72小时,并使用RT-PCR和RT-qPCR评估COL1A1和CEMIP基因表达,无论是在整个组织碎片中还是在通过激光捕获显微切割分离的肌成纤维细胞中。
结果:从腹膜(PER)获得的子宫内膜异位组织碎片的代谢活性,卵巢(OMA),深(DE)子宫内膜异位病灶在基于二甲基亚砜的培养基中冷冻保存后保存良好,与新鲜组织碎片相当。与新鲜组织相比,PER的相对代谢活性为70%(CI:92-47%),43%(CI:53-15%)在OMA和94%(CI:186-3%)在DE病变。在来自PE病变的碎片中,92%(CI:87-96%),OMA病变95%(CI:91-98%),来自DE病变的88%(CI:78-98%)的细胞在冷冻保存和解冻后24小时培养期后存活。在用吡非尼酮或二甲双胍治疗72小时后,可以在整个组织片段和分离的肌成纤维细胞中检测到纤维化标志物COL1A1和CEMIP的基因表达差异,表明冷冻保存和解冻的子宫内膜异位组织碎片适用于测试抗纤维化干预措施。
方法:不适用。
结论:子宫内膜异位组织碎片的活力和代谢活性可能因外科手术过程中的损伤而部分受损,导致样本间方差。
结论:保存活的子宫内膜异位组织碎片供以后在离体模型中使用,为有效测试潜在的新治疗策略创造了可能性,并促进了不同研究实验室之间活的子宫内膜异位组织的交换。
背景:这项研究没有外部资金支持。作者宣布没有竞争利益。
背景:不适用。
OBJECTIVE: Is it possible to establish an ex vivo endometriosis model using cryopreserved endometriotic tissue fragments?
CONCLUSIONS: Cryopreserved endometriotic tissue fragments remain viable after thawing and during at least 3 days of culture and can therefore be used to establish an ex vivo endometriosis model to efficiently test potential therapeutic agents.
BACKGROUND: Endometriosis is the most prevalent benign gynecologic disease with an enormous societal burden; however, curative therapies are still lacking. To efficiently test potential new therapies, an ex vivo model based on previously cryopreserved endometriotic tissue that recapitulates the different endometriosis subtypes and their microenvironment is highly desirable.
METHODS: Endometriotic tissue fragments of three different subtypes were obtained from 28 patients by surgical resection. After cryopreservation and thawing, viability and metabolic activity of these tissue fragments were assessed. Viability was compared with fresh fragments from 11 patients directly after surgical removal. Experimental intervention studies were performed in cryopreserved and thawed tissue fragments from two patients to confirm the usability of these tissues for ex vivo intervention studies.
METHODS: Endometriotic tissue fragments (n = 45) were cryopreserved according to three different protocols. After thawing, fragments were cultured for 24 h. A resazurin-based assay was performed to assess the metabolic activity of the tissue fragments. In addition, cell type-specific viability was analyzed by VivaFix, Hoechst 33342, and α-smooth muscle actin immunofluorescence staining and confocal microscopy. The presence of endometriosis was histologically confirmed based on hematoxylin-eosin staining. Cryopreserved and thawed tissue fragments were treated for 72 h with pirfenidone or metformin and COL1A1 and CEMIP gene expressions were assessed using RT-PCR and RT-qPCR, either in the whole tissue fragments or in myofibroblasts isolated by laser capture microdissection.
RESULTS: Metabolic activity of endometriotic tissue fragments obtained from peritoneal (PER), ovarian (OMA), and deep (DE) endometriotic lesions was well preserved after cryopreservation in a dimethyl sulfoxide-based medium and was comparable with fresh tissue fragments. Relative metabolic activity compared to fresh tissue was 70% (CI: 92-47%) in PER, 43% (CI: 53-15%) in OMA and 94% (CI: 186-3%) in DE lesions. In fragments from PE lesions 92% (CI: 87-96%), from OMA lesions 95% (CI: 91-98%), and from DE lesions 88% (CI: 78-98%) of cells were viable after cryopreservation and thawing followed by a 24-h culture period. Differences in gene expression of fibrotic markers COL1A1 and CEMIP after 72-h treatment with pirfenidone or metformin could be detected in whole tissue fragments and in isolated myofibroblasts, indicating that cryopreserved and thawed endometriotic tissue fragments are suitable for testing anti-fibrotic interventions.
METHODS: N/A.
CONCLUSIONS: Viability and metabolic activity of the endometriotic tissue fragments may have been partially compromised by damage sustained during the surgical procedure, contributing to inter-sample variance.
CONCLUSIONS: The storage of viable endometriotic tissue fragments for later usage in an ex vivo model creates the possibility to efficiently test potential new therapeutic strategies and facilitates the exchange of viable endometriotic tissue between different research laboratories.
BACKGROUND: This study was not financially supported by external funding. The authors declare no competing interest.
BACKGROUND: N/A.