A protocol to isolate and qualify purified human preantral follicles in cases of acute leukemia, for future clinical applications

Abstract

BackgroundAutotransplantation of cryopreserved ovarian cortex can be associated with a risk of cancer cell reseeding. This issue could be eliminated by grafting isolated preantral follicles. Collagenase NB6 is an enzyme produced under good manufacturing practices (GMP) in compliance with requirements for tissue engineering and transplantation in humans and thus can be used to isolate preantral follicles from ovarian tissue in the framework of further clinical applications. Multicolor flow cytometry is an effective tool to evaluate the potential contamination of follicular suspensions by leukemic cells.MethodsThe efficiency of collagenase NB6 was evaluated in comparison to collagenase type IA and Liberase DH, in terms of yield, morphology and viability. A short-term in vitro culture of follicles isolated with collagenase NB6 was conducted for 3 days in a fibrin matrix. A modelization procedure was carried out to detect the presence of leukemic cells in follicular suspensions using multicolor flow cytometry (MFC).ResultsNo statistical differences were found between collagenase NB6, Liberase DH (p = 0.386) and collagenase type IA (p = 0.171) regarding the number of human preantral follicles isolated. The mean diameter of isolated follicles was significantly lower with collagenase NB6 (p < 0.0001). The survival rate of isolated follicles was 93.4% (n = 272) using collagenase NB6 versus 94.9% (n = 198) with Liberase DH and 92.6% (n = 298) using collagenase type IA. Even after 3 days of in vitro culture in a fibrin scaffold, most of the isolated follicles were still alive after using collagenase NB6 (90.7% of viable follicles; n = 339). The rate of isolated Ki67-positive follicles was 29 ± 9.19% before culture and 45 ± 1.41% after 3 days. In 23 out of 24 follicular suspensions analyzed, the detection of leukemic cells by MFC was negative. The purification had no significant impact on follicle viability.ConclusionThe isolation and purification of human preantral follicles were performed following good manufacturing practices for cell therapy. Multicolor flow cytometry was able to confirm that final follicular suspensions were free from leukemic cells. This safe isolation technique using collagenase NB6 can be considered for future clinical applications.

Bioengineering of Human Artificial Ovary From Cryopreserved Tissue: Optimization of the Follicles Enzymatic Isolation.

“…Follicles diameter in Group 1 has signi cantly smaller than Group 2 (44.5 ± 11.4 μm vs 45.8 ± 11.1 μm, p < 0.01). This were similar to the result obtained by Mouloungui et al who reported the similar mean diameter of follicles treatment by Liberase DH (45.2 ± 8.2 μm) [27].…”

Bioengineering anin situovary (ISO) for fertility preservation

“…A major benefit of isolating follicles from the ovarian stroma is the potential to reduce any malignant cells that could be introduced during transplantation. Although we did not validate this with our isolation methods, there have been reports suggesting that follicle isolation can significantly decrease cancer cells prior to implantation 15,69 . In addition, our isolated ovarian cell suspensions were not filtered to contain follicles alone.…”

Fertility Preservation: The Challenge of Freezing and Transplanting Ovarian Tissue

“…In view of this risk, various research teams worldwide have been investigating alternative approaches, including: (i) in vitro (ex vivo) culture of follicles [122][123][124], and (ii) in vivo (post-transplantation) growth of isolated preantral follicles inside a transplantable artificial ovary [92,[125][126][127][128][129][130][131][132][133][134][135][136][137][138][139][140]. The primary objective of an artificial ovary is to safely transplant ovarian follicles, while ensuring secretion of sex hormones and development of fertilizable mature oocytes.…”