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

BACKGROUND: Autotransplantation 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. METHODS: The 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). RESULTS: No 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. CONCLUSION: The 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.

Validation of an automated technique for ovarian cortex dissociation: isolation of viable ovarian cells and their qualification by multicolor flow cytometry.

BACKGROUND: Ovarian tissue cryopreservation is a technique for fertility preservation addressed to prepubertal girls or to patients for whom no ovarian stimulation is possible before initiation of gonadotoxic treatments. Autotransplantation of frozen-thawed ovarian tissue is the only available option for reuse but presents some limitations: ischemic tissue damages post-transplant and reintroduction of malignant cells in cases of cancer. It is therefore essential to qualify ovarian tissue before autograft on a functional and oncological point of view. Here, we aimed to isolate viable cells from human ovarian cortex in order to obtain an ovarian cell suspension analyzable by multicolor flow cytometry. METHODS: Ovarian tissue (fresh or frozen-thawed), from patients with polycystic ovarian syndrome (reference tissue) and from patients who underwent ovarian tissue cryopreservation, was used for dissociation with an automated device. Ovarian tissue-dissociated cells were analyzed by multicolor flow cytometry; the cell dissociation yield and viability were assessed. Two automated dissociation protocols (named laboratory and commercial protocols) were compared. RESULTS: The effectiveness of the dissociation was not significantly different between reference ovarian tissue (1.58 × 106 ± 0.94 × 106 viable ovarian cells per 100 mg of ovarian cortex, n = 60) and tissue from ovarian tissue cryopreservation (1.70 × 106 ± 1.35 × 106 viable ovarian cells, n = 18). However, the viability was slightly different for fresh ovarian cortex compared to frozen-thawed ovarian cortex whether we used reference tissue (p = 0.022) or tissue from ovarian cryopreservation (p = 0.018). Comparing laboratory and commercial protocols, it appeared that cell yield was similar but cell viability was significantly improved when using the commercial protocol (81.3% ± 12.3% vs 23.9% ± 12.5%). CONCLUSION: Both dissociation protocols allow us to isolate more than one million viable cells per 100 mg of ovarian cortex, but the viability is higher when using the commercial dissociation kit. Ovarian cortex dissociation is a promising tool for human ovarian cell qualification and for ovarian residual disease detection by multicolor flow cytometry.

Tubular Compressed Collagen Scaffolds for Ureteral Tissue Engineering in a Flow Bioreactor System.

Ureteral replacement by tissue engineering might become necessary following tissue loss after excessive ureteral trauma, after retroperitoneal cancer, or even after failed reconstructive surgery. This need has driven innovation in the design of novel scaffolds and specific cell culture techniques for urinary tract reconstruction. In this study, compressed tubular collagen scaffolds were evaluated, addressing the physical and biological characterization of acellular and cellular collagen tubes in a new flow bioreactor system, imitating the physiological pressure, peristalsis, and flow conditions of the human ureter. Collagen tubes, containing primary human smooth muscle and urothelial cells, were evaluated regarding their change in gene and protein expression under dynamic culture conditions. A maximum intraluminal pressure of 22.43 ± 0.2 cm H2O was observed in acellular tubes, resulting in a mean wall shear stress of 4 dynes/cm(2) in the tubular constructs. Dynamic conditions directed the differentiation of both cell types into their mature forms. This was confirmed by their gene expression of smooth muscle alpha-actin, smoothelin, collagen type I and III, elastin, laminin type 1 and 5, cytokeratin 8, and uroplakin 2. In addition, smooth muscle cell alignment predominantly perpendicular to the flow direction was observed, comparable to the cell orientation in native ureteral tissue. These results revealed that coculturing human smooth muscle and urothelial cells in compressed collagen tubes under human ureteral flow-mimicking conditions could lead to cell-engineered biomaterials that might ultimately be translated into clinical applications.