Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe

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Cryopreservation and CAM-xenotransplantation of human ovarian tissue

We would like to call your attention to the following One-on-One Training Course (hands-on workshop) on Cryopreservation and CAM-xenotransplantation of human ovarian tissue. This workshop will provide exclusive face-to-face individual training to facilitate learning, and the approach is anticipated to provide value-for-cost for participants as this type of training is more effective than group training. We extend a cordial invitation to you to attend this event and will be pleased to welcome you to Cologne.

Generally about the workshop

This workshop is intended for postgraduate students and specialists in the field of cryopreservation of human reproductive cells.
Our course contains short basic lectures and films regarding the technology. The central part of our workshop is hands-on manipulations.
All demonstrating manipulations will be performed on bovine ovarian tissue. 

A few concise paragraphs on technologies and the workshop 

Each element of the technology is a result of our own research that has already been published [1–38]. In the description of the course we present the respective articles in squire brackets.
The organizers of this course transplanted ovarian tissue that had been cryo-stored for five years and achieved a pregnancy and a baby—the first in Germany [22, 23]. We will present the technique of ovarian tissue cryopreservation modernized over the last years.

This course will include three major parts:

  • The first part: Technology of low-temperature transportation, freezing and thawing of ovarian tissue [6–28, 31, 34, 35, 37].
  • The second part: Technology of cryopreservation of whole human ovaries with pedicles [29, 30, 32, 33]. The near future: Construction of human artificial ovaries from cryopreserved ovarian tissue [36, 38].
  • The third part: Technology for CAM-xenotransplantation of thawed ovarian tissue [21, 24, 25, 27, 28].

Course directors

  • Prof. Dr. Peter Mallmann, Director of University Maternal Hospital
  • Dr. (SU) Evgenia Isachenko, Biological Director of IVF-Laboratory
  • Prof. Dr. Gohar Rahimi, Medical Director of IVF-Laboratory
  • Prof. Dr. Raul Sanchez, Director of Biomedical Center, La Frontera University, Temuco, Chile (during certain periods only)
  • Dr. (SU) Vladimir Isachenko, Leader of Research Group for Reproductive Medicine 

Further Information and Program

Date: Beginning October 2019 (any date at the participant’s convenience)
Duration: 3 days
Number of participants: One, a maximum of two per session
Location: Cologne University Maternal Hospital, Research Laboratory for Reproductive Medicine, Kerpener Str. 34, 50931 Cologne, Germany
Language: English and German
Course fee: 2500 EUR for a single participant, 3000 EUR for two participants

Information and registration:
Dr. (SU) Volodimir Isachenko
Phone +49 221 478-7349
E-Mail vladimir.isachenko@uk-koeln.de

About the place
Cologne is a famous tourist city. Staying in Cologne allows easy access to the Netherlands, Belgium, Luxemburg and France. For example, a tour to Amsterdam with a 5 to 6 hour stay in the city with transport Cologne-Amsterdam-Cologne occupies a little bit more than one light-day and costs up to 25 EUR. A 4-day tour to Paris (3 days in this city) including transport und hotel costs from 90 to 180 EUR.

Program

First day
TimeProgramCourse director
08:00-08:45Lecture with discussion
Technology of 24 hours low-temperature transportation, preparation and freezing of ovarian tissue of Patient W.
the first baby born in Germany after cryopreservation of ovarian tissue): detailed information [22, 23].  		Dr. (SU) Evgenia Isachenko
09:00-09:45Lecture with discussion
Modernized technology for ovarian tissue as a result of 14 years of research [6–21, 24–28, 31, 34, 35].		Dr. (SU) Vladimir Isachenko
10:00-10:45Film with comments and discussion
Year 2018. Modernized technology of preparation, freezing, thawing and in vitro culture of human ovarian tissue. 		Dr. (SU) Vladimir Isachenko
11:00-12:45 and
13:10-16:00		Hands-on
  • Long-term transport of ovarian tissue [15, 26, 27, 34].
  • Removal of GV-Oocytes (puncture of follicles) in fresh ovarian tissue before cooling as element of concept of our cryobank.
  • Preparation of strips of ovarian tissue: release of cortex from medulla [24, 35].
  • One-day cooling of ovarian tissue as an important element of cryopreservation protocol [27, 34].
  • Saturation of tissue with cryoprotectants on the moving system [6].
  • Freezing of strips in 5 ml Cryo-Vials with different freezing constructions; the role of ice-seeding [8].
 

Second day
TimeProgramCourse director
08:00-08:45Lecture with discussion
Technology of cryopreservation of whole human ovaries with pedicles.
The near future: Construction of a human artificial
ovary from cryopreserved ovarian tissue [29, 30, 32, 33, 37, 38].  		Prof. Dr. Peter Mallmann
09:00-12:50 and
13:10-16:00		Hands-on
  • Quick thawing of ovarian tissue in boiling water and removal of cryoprotectants [12, 14, 17]
  • In vitro culture of ovarian tissue in large volume with agitation [6].
  • Evaluation of viability of ovarian tissue: quality of follicles as central criterion of this evaluation [18].
  • Storage and culture of chicken eggs.
  • Manufacturing and preparation of silicon rings.
  • Drilling of eggs and cutting a window.
  • Placing the silicon rings on the CAM surface and transfer of tissues and tumor cells.
Dr. (SU) Evgenia Isachenko
Prof. Dr. Gohar Rahimi
13:10-14:10Film with comments and discussion
Technology of CAM-xenotransplantation of ovarian, testicular and cancer tissues [21, 24, 25, 27, 28]. 		Dr. (SU) Vladimir Isachenko
Third day
TimeProgramCourse director
09:00-10:00Lecture with discussion
Cryopreservation of human ovarian tissue: freezing vs. vitrification [2-5, 10, 12, 13, 14, 16, 17,19].		 

10:00-12:50 and

13:10-16:00

Hands-on
  • Extraction of tissues after culture and fixation for evaluation of their viability.
  • Evaluation of angiogenesis during CAM-Culture of tissues [26].
  • Injection of solutions into blood capillaries.
  • Macroscopic and microscopic examination of CAM-cultured tissues.
  • SCID-Mouse-Xenotransplantation of ovarian tissue [5, 13, 17, 34, 35].   

Prof. Dr. Gohar Rahimi
Dr. (SU) Evgenia Isachenko
Dr. (SU) Vladimir Isachenko

16:00-17:00Extensive discussion and repeated hands-on manipulationsProf. Dr. Peter Mallmann
Dr. (SU) Evgenia Isachenko
Dr. (SU) Vladimir Isachenko
Prof. Dr. Gohar Rahimi
17:00-17:15Presentation of certificates and departure 

Publications

Our publications are mentioned in this announcement

[1] Measurement of apoptosis in long-term cultures of human ovarian tissue. Reproduction (2001) 
[2] Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: negative effect of disaccharides in vitrification solution. CryoLetters (2002) 
[3] Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen. Eur. J. Obstetr. Gyn. R.B. (2003) 
[4] Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis. Reprod. Fert. Develop. (2003) 
[5] Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice. Reprod. Biomed. Online (2004)
[6] Effective method for in-vitro culture of cryopreserved human ovarian tissue. Reprod. Biomed. Online (2006) 
[7] Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing. Cryobiology (2007) 
[8] Cryopreservation of human ovarian tissue: effect of spontaneous and initiated ice formation. Reprod. Biomed. Online (2008) 
[9] Simplified technique of human ovarian tissue freezing: quick cooling from -36 degree C. CryoLetters (2008) 
[10] Human ovarian tissue preservation: is vitrification acceptable method for assisted reproduction? CryoLetters (2008) 
[11] Eine Kryobank für humanes Ovarialgewebe: Konzept und Perspektiven. Frauenarzt (2008) 
[12] Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction (2009) 
[13] Apoptosis in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. CryoLetters (2009) 
[14] Human ovarian tissue: vitrification versus conventional freezing. Hum. Reprod. (2009) 
[15] Effect of long-term exposure at suprazero temperatures on activity and viability of human ovarian cortex. Fertil. Steril. (2009) 
[16] Cryobanking of human ovarian tissue for anti-cancer treatment: comparison of vitrification and conventional freezing. CryoLetters (2009) 
[17] Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. Eur. J. Obstet. Gyn. R. B. (2010) 
[18] Human ovarian tissue cryopreservation: quality of follicles as a criteria of effectiveness. Reprod. Biomed. Online (2010) 
[19] Genexpression und Morphologie der Follikel nach konventionellem Einfrieren und Vitrifikation von humanem Ovarialgewebe. Geburtsh. Frauenheilk. (2010) 
[20] Concept of human ovarian tissue cryobanking. In: R.-Ch. Chian, P. Quinn, eds. “Fertility cryopreservation”, Cambridge University Press, New York, USA (2010)
[21] Vascularised chorioallantoic membrane (CAM) culture system for cryopreserved human ovarian tissue as an alternative to xenotransplantation). Geburtsh. Frauenheilk. (2011)  
[22] First live birth in Germany after re-transplantation of cryopreserved human ovarian tissue: original device for initiated ice-formation. Clin. Lab. (2012) 
[23] Cryopreservation of ovarian tissue: detailed description of methods for transport, freezing and thawing. Geburth. Frauenheilk. (2012) 
[24] Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue. PLoS One (2012)
[25] Die Chorioallantoismembran des Huhns Kultursystem für kryokonserviertes menschliches Ovarialgewebe (The chicken chorioallantoic membrane. A culture system for cryopreserved human ovarian tissue). Gynaekol. Endokrinol. (2012) 
[26] Long-time cooling of human ovarian tissue before cryopreservation as obvious procedure: stimulation of follicular development and neo-vascularisation. Clin. Lab. (2012) 
[27] Increasing follicular and stromal cell proliferation in cryopreserved human ovarian tissue after long-term precooling prior to freezing: in vitro versus  chorioallantoic membrane (CAM) xenotransplantation. Cell Transplant. (2013) 
[28] Viability of human ovarian tissue confirmed 5 years after freezing with spontaneous ice-formation by autografting and chorio-allantoic membrane culture. Cryobiology (2013)
[30] In vitro perfusion of whole bovine ovaries by freezing medium: effect of perfusion rate and elapsed time after extraction. Clin. Lab. (2013)  
[31] Whole ovine ovaries as a model for human: perfusion with cryoprotectants in vivo and in vitro. Biomed. Res. Int. (2014) 
[32] Transplantation of cryopreserved human ovarian tissue: restoration of reproductive function after two cycles of radio- and chemo-therapeutic treatments. Clin. Lab. (2014) 
[33] Whole ovine ovaries as a model for human: perfusion with cryoprotectants in vivo and in vitro. Biomed. Res. Int. (2014) 
[34] Cryopreservation of whole ovine ovaries with pedicles as a model for human: parameters of perfusion with simultaneous saturations by cryoprotectants. Clin. Lab. (2015)
[35] Long-time cooling before cryopreservation decreased translocation of phosphatidylserine (Ptd-L-Ser) in human ovarian tissue. PLoS One (2015)
[36] Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate. Reprod. Biol. Endocrinol. (2016)
[37] Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex. Reprod. Biol. Endocrinol. (2018) 
[38] A successful multidisciplinary approach for treatment and for preserving the reproductive potential in a rare case of acute lymphocytic leukemia during pregnancy. Gynecol. Endocrinol. (2018) 
[39] Construction of human artificial ovary from cryopreserved ovarian tissue: Appearance of apoptosis and necrosis after enzymatic isolation of follicles. Cryobiology (2018)

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Set for freezing of ovarian tissue. Source: Uniklinik Köln

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Operative removal of ovarian fragments. Source: Uniklinik Köln

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Partial removal of medulla from cortex. Source: Uniklinik Köln

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Partial removal of medulla from cortex. Source: Uniklinik Köln

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Puncture of GV-Oocytes before cooling. Source: Uniklinik Köln

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Location of ovarian tissue to cryo-vial with freezing medium. Source: Uniklinik Köln

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Location of ovarian tissue to cryo-vial with freezing medium. Source: Uniklinik Köln

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Ovarian tissue in cryo-vial just before cooling. Source: Uniklinik Köln

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Artificial ice formation (seeding). Source: Uniklinik Köln

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Thawing of ovarian tissue. Source: Uniklinik Köln

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Step-wise rehydration of ovarian tissue. Source: Uniklinik Köln

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CAM-Xenotransplantation: opening of chicken egg. Source: Uniklinik Köln

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CAM-Xenotransplantation: opening of chicken egg. Source: Uniklinik Köln

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CAM-Xenotransplantation: transfer of cells suspension. Source: Uniklinik Köln

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CAM-Xenotransplantation: cells after culture. Source: Uniklinik Köln

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CAM-Xenotransplantation: cells after culture. Source: Uniklinik Köln

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CAM-Xenotransplantation: ovarian piece after culture. Source: Uniklinik Köln

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CAM-Xenotransplantation: ovarian piece after culture. Source: Uniklinik Köln

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CAM-Xenotransplantation: ovarian piece after culture. Source: Uniklinik Köln

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