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Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue.

Isachenko V, Mallmann P, Petrunkina AM, Rahimi G, Nawroth F, Hancke K, Felberbaum R, Genze F, Damjanoski I, Isachenko E - PLoS ONE (2012)

Bottom Line: Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture).It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Cologne University, Cologne, Germany. v.isachenko@yahoo.com

ABSTRACT
At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.

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Cryopreserved ovarian medulla-free and medulla-containing pieces before and after 5 days culture with chorioallantoic membrane (CAM) system.(a, b, c, d, e) medulla-free piece, (a, b) just after thawing and seeding on CAM marked by silicone ring, (c, d, e) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (f, g, h, i, j) medulla-containing piece, (f, g) just after thawing and seeding on CAM marked by silicone ring, (h, i, j) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (e) outer CAM-layer with medulla-free piece, (j) inner CAM-layer with medulla-containing piece. Different intensiveness of the avian vascularisation in the place of the seeding of pieces was noted: (e) versus (j). Bar = 1 mm.
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pone-0032549-g002: Cryopreserved ovarian medulla-free and medulla-containing pieces before and after 5 days culture with chorioallantoic membrane (CAM) system.(a, b, c, d, e) medulla-free piece, (a, b) just after thawing and seeding on CAM marked by silicone ring, (c, d, e) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (f, g, h, i, j) medulla-containing piece, (f, g) just after thawing and seeding on CAM marked by silicone ring, (h, i, j) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (e) outer CAM-layer with medulla-free piece, (j) inner CAM-layer with medulla-containing piece. Different intensiveness of the avian vascularisation in the place of the seeding of pieces was noted: (e) versus (j). Bar = 1 mm.

Mentions: Fertilized eggs of White Leghorn chickens, purchased at a local hatchery and incubated at 37°C with 60% relative humidity, were prepared for implantation on day 4 of incubation. Standard microbiology assessment was performed to exclude subclinical infections. Preparation of the chorio-allantoic membranes was performed essentially as previously described [50], [54]–[56]. Each egg was washed with warm 70% ethanol, after which a hole was drilled through the pointed pole of the shell. The following day, part of the chorio-allantoic membrane of the embryo was exposed by peeling a 1.5–2.0 cm window in the shell. This window was covered with tape and the incubation continued. The chorio-allantantoic membrane has two epithelial layers and, in its intact form, represents a “dry” impermeable barrier for all invasions, including ovarian fragments. For “connection” of ovarian pieces with the egg system, the latter must be open. To this end, the upper peridermal part of the double epithelial layer was removed in each egg, leaving the basal layer intact. On day 10 of incubation a silicone ring 0.5 mm thick with a 5 mm inner diameter was placed on the membrane. An ovarian piece was placed into this silicone ring using microsurgical forceps (Fig. 2). Thereafter, the shell window was covered again, and the egg replaced in the incubator. After 5 days of CAM-culture the survival rate of the ovarian piece was evaluated.


Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue.

Isachenko V, Mallmann P, Petrunkina AM, Rahimi G, Nawroth F, Hancke K, Felberbaum R, Genze F, Damjanoski I, Isachenko E - PLoS ONE (2012)

Cryopreserved ovarian medulla-free and medulla-containing pieces before and after 5 days culture with chorioallantoic membrane (CAM) system.(a, b, c, d, e) medulla-free piece, (a, b) just after thawing and seeding on CAM marked by silicone ring, (c, d, e) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (f, g, h, i, j) medulla-containing piece, (f, g) just after thawing and seeding on CAM marked by silicone ring, (h, i, j) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (e) outer CAM-layer with medulla-free piece, (j) inner CAM-layer with medulla-containing piece. Different intensiveness of the avian vascularisation in the place of the seeding of pieces was noted: (e) versus (j). Bar = 1 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316540&req=5

pone-0032549-g002: Cryopreserved ovarian medulla-free and medulla-containing pieces before and after 5 days culture with chorioallantoic membrane (CAM) system.(a, b, c, d, e) medulla-free piece, (a, b) just after thawing and seeding on CAM marked by silicone ring, (c, d, e) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (f, g, h, i, j) medulla-containing piece, (f, g) just after thawing and seeding on CAM marked by silicone ring, (h, i, j) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (e) outer CAM-layer with medulla-free piece, (j) inner CAM-layer with medulla-containing piece. Different intensiveness of the avian vascularisation in the place of the seeding of pieces was noted: (e) versus (j). Bar = 1 mm.
Mentions: Fertilized eggs of White Leghorn chickens, purchased at a local hatchery and incubated at 37°C with 60% relative humidity, were prepared for implantation on day 4 of incubation. Standard microbiology assessment was performed to exclude subclinical infections. Preparation of the chorio-allantoic membranes was performed essentially as previously described [50], [54]–[56]. Each egg was washed with warm 70% ethanol, after which a hole was drilled through the pointed pole of the shell. The following day, part of the chorio-allantoic membrane of the embryo was exposed by peeling a 1.5–2.0 cm window in the shell. This window was covered with tape and the incubation continued. The chorio-allantantoic membrane has two epithelial layers and, in its intact form, represents a “dry” impermeable barrier for all invasions, including ovarian fragments. For “connection” of ovarian pieces with the egg system, the latter must be open. To this end, the upper peridermal part of the double epithelial layer was removed in each egg, leaving the basal layer intact. On day 10 of incubation a silicone ring 0.5 mm thick with a 5 mm inner diameter was placed on the membrane. An ovarian piece was placed into this silicone ring using microsurgical forceps (Fig. 2). Thereafter, the shell window was covered again, and the egg replaced in the incubator. After 5 days of CAM-culture the survival rate of the ovarian piece was evaluated.

Bottom Line: Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture).It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Cologne University, Cologne, Germany. v.isachenko@yahoo.com

ABSTRACT
At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.

Show MeSH
Related in: MedlinePlus