Day 1 :
The Andrology Institute of America, USA
Time : 9:45-10:10
M Zavos received his BS in Biology-Chemistry in 1970, his MS in Biology-Physiology in 1972 and Education Specialist in Science (EdS) in 1976 from Emporia State University in Emporia, Kansas. He earned his PhD in Reproductive Physiology, Biochemistry and Statistics in 1978 from the University of Minnesota in the Twin Cities, Minnesota. He received the distinguished Alumnus Award and the Graduate Teaching Award from Emporia State University and the Student Leadership Award from the University of Minnesota. He has numerous scientific collaborations nationally and internationally and his publications have appeared in eight languages. He is a Member of the American Society for Reproductive Medicine (ASRM), the American Society of Andrology (ASA), and the European Society for Human Reproduction and Embryology (ESHRE), the Middle East Fertility Society (MEFS), the Japanese Fertility Society, the International Society of Cryobiology Sigma XI, Gamma Sigma Delta and a number of other Scientific and Professional Societies. He has served on a large number of committees for the International Society of Cryobiology, ASRM, MEFS, ESHRE and others.
Semen or sperm cryopreservation (commonly called sperm banking) is a procedure to preserve sperm cells. Semen can be used successfully indefinitely after cryopreservation which normally is kept at a very stable temperature of -196ºC (liquid Nitrogen). For human sperm, the longest reported successful storage which yielded a successful pregnancy is 22 years. The cryopreservation procedure can be used for sperm donation where the recipient wants the treatment in a different time or place, or as a means of preserving fertility for men undergoing vasectomy or other treatments that may compromise their fertility, such as chemotherapy, radiation therapy or surgery. Cryopreservation extends the availability of sperm for fertilization; however, the fertilizing potential of the frozen-thawed sperm is compromised because of alterations in the structure and physiology of the sperm cell. These alterations, characteristics of sperm capacitation, are present in the motile population and decrease sperm life-span, ability to interact with the female tract, and subsequent fertilizing ability. The etiology of such alterations may represent a combination of factors, such as inherited sensitivity of the sperm cell to withstand the cryopreservation process and the semen dilution. Although the former is difficult to address, approaches that make-up for the dilution of seminal fluid may be sought. The aim of this work is to review aspects of sperm cryopreservation paralleled by events of capacitation and evaluate the possible roles of sperm membrane cholesterol, reactive oxygen species, and seminal plasma as mediators of cryopreservation effects on sperm function. As far as the methods of cryopreservation of human sperm, there are three main conventional freezing techniques used in sperm cryopreservation: Slow freezing, rapid freezing and vitrification. The slow freezing technique which consists of progressive sperm cooling over a period of 2–4 hours in two or three steps, either manually or automatically using a semi programmable freezer; The rapid freezing technique which requires direct contact between the straws and the nitrogen vapors for 8–10 min and immersion in liquid nitrogen at −196°C and lastly, the vitrification method which is the newest procedure to be followed and which renders very high cooling and warming rates (over 20,000°C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over −180°C) and which also offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. All three of those methods are clinically employed and some of their technical aspects will be evaluated and compared along with their implications on the outcome of clinical results.
Fraunhofer Institute for Biomedical Engineering, Germany
Keynote: Improved methods and procedures for pluripotent stem cell preservation, storage stability and validation
Time : 10:10-10:35
Heiko Zimmermann is the Director and Head of Fraunhofer IBMT and Chair in Molecular and Cellular Biotechnology at the Saarland University and has been working as Physicist since 1997 in the field of cell biophysics. He coordinated the EU-project HYPERLAB in FP7 and was WP leader in several other EU-projects. Within EBiSC, he is Leader of WP 3 and is additionally coordinating the translational project DropTech® in FP7. He received the first permission for working with hESCs within the Fraunhofer Gesellschaft. He is the author of more than 70 peer-reviewed papers and book chapters. His research expertise covers cryobiology, cryotechnology and biopolymers for clinical scaffolds. He is inventor of more than 50 granted patent families from which more than 20 have been commercially licensed.
The project EBiSC aims to build up a European biobank for research grade human induced pluripotent stem cells (hIPSCs). The vision of EBiSC leads to the demand for upscaled production methods, these kinds of cells leading to the need for automated systems and procedures in stem cell processing and banking. An overview of existing state-of-the-art automation systems is given and the specifications for different applications are compared. Furthermore, modules and concepts for automated cell identification, pluripotency testing, and viability and functionality tests are drawn and results are shown. Scalable label-free analysis of pluripotent stem cells using quantitative life cell imaging and on-line image analysis is shown. A specialized system, the automated hanging drop technique (DropTech®) is shown. The DropTech system allows fully automated cultivation of hiPSCs on micro carrier using the hanging drop technology and enables applications like the automated Embryonic Stem cell Test (EST) for standardized embryo toxicity tests. The last part of the talk deals with the technology of cryopreservation, banking and validation frozen samples. The method of surface-based vitrification of pluripotent stem cells is introduced and the need for a completely closed cool chain is derived from experimental results. Solutions for automated industrial scale biobanking with closed cool chains and with minimal harmful thermal fluctuations are shown and the effect on functionality of cryopreserved cells compared to standard technology is shown. A method for non-invasive monitoring of re-crystallization and de-vitrification effects using Raman micro-spectroscopy is presented.
Belgorod National Research University, Russia
Time : 10:35-11:00
Igor I Katkov is a trained Biophysicist with more than 30 years of experience in Cryobiology and Cryogenic Engineering. His research has been focused on the fundamental aspects of kinetic vitrification (K-VF) as well as on designing the practical system for K-VF KrioBlast™ in cooperation with V F Bolyukh from Ukraine. He is a Chief Scientific Officer of Celltronix, San Diego, USA and recently accepted a Professor-level position of the Head of the Laboratory of Amorphous State in the Belgorod National Research University, Russia.
Cryopreservation (CP) and subsequent long-term storage (cryobanking) are important parts of both life science research and related industries and technologies. As we have stated before, there are 5 basics ways of achieving long-term storage, all essentially lead to vitrification of cells, namely: Slow freezing (SF), Equilibrium vitrification (E-VF) with high concentration of vitrificants (“thickeners”) and relatively moderate speed of cooling and rewarming, Kinetic vitrification (K-VF) with very rapid rates of cooling and rewarming and low to none concentration of exogenous vitrificants, Freeze-drying (lyophilization), and Vacuum/air flow drying at temperatures above zero degree Celsius (xeropreservation), which up to now, is the mainstream of the majority of CP technologies. It however requires multi-step protocols, expensive programmable freezers and must be tuned to the particular types of cells, tissues and organs. In this presentation, we will focus on the kinetic vs. equilibrium vitrification. We will compare the mechanisms, analyze phase diagrams, emphasize the role of the Leidenfrost effect (LFE) and ways of reducing up to full eliminating LFE, discuss pros and cons of each methods and present information on basic equipment and accessories for E-KF and K-VF used in different field of biology, reproductive, regenerative and personalized medicine, drug screening, agriculture, conservation of endangered species, medicine and other related disciplines of sciences and industries. And, finally, we will introduce our (solution to hyper-fast cooling and present a short video clip of the novel hyper-fast scalable cooling devices KrioBlast™ and VitriPlunger™ and briefly discuss the promising results on K-VF of human sperm, embryonic stem cells and insulin-producing cells using the KrioBlas™ and VitriPlunger™ systems.
GenLife Institute for Regenerative Medicine and Stem Cells, USA
Time : 11.20-11:45
Charles F Mahl is a graduate of Case Western Reserve University in Cleveland, Ohio. He has received his MD degree from the Rosalind Franklin School of Medicine in Chicago, Illinois. He did his Residency and Chief Residency in New York at Interfaith Hospital and Vitreo Retinal Fellowship at the University of Oregon. He has completed a Fellowship program in Anti-Aging and Regenerative Medicine by the American Academy of Anti-Aging Medicine, the Living Younger Preventive Aging Program and is certified in Age Management Medicine by The Cenegenics Education and Research Foundation. He is Regenestem trained in both PRP and adipose/bone marrow derived stem cells. He is Board Certified in Integrative Medicine (BCIM) and is a Certified Sports Trainer (CST) and Certified Sports Nutritionist (CSN). He is also a Member of the Advisory Board of Global Stem Cells and Regenestem.
Prolotherapy has been around for almost 80 years, yet is not well known in most musculoskeletal circles. Most orthopedic surgeons and neurosurgeons either have not ever heard of prolotherapy or think it does not work based on never having studied it in residency training programs, their personal bias or their general lack of knowledge about it. The author will explain his discovery of prolotherapy after some 30 years of suffering from pain and his experience and will introduce this fine medical art to the participants. Prolotherapy was the first procedure invented in Regenerative Medicine, followed by PRP-Platelet Rich Plasma injections and then Stem Cell Injections from the bone marrow and/or adipose tissue. Prolotherapy is a simple technique, much less invasive and less harmful as compared to steroid injections and high risk musculoskeletal surgery and often times, when performed by a skilled and experienced prolotherapist can alleviate the chronic pain one is suffering from. The author will explain the physiology of ligaments and tendons in response to prolotherapy and differentiate between 1st line, 2nd line and 3rd line regenerative medicine therapies. Prolotherapy is the first line of defense when it comes to regenerative medicine and should be recommended to patients prior to cortisone injections or invasive surgery. Elite athletes are already aware of its potential and utilize it when injured and or reinjured to heal their painful joints. It is time for prolotherapy to become main stream and no longer be considered alternative medicine. Author will describe the indications for prolotherapy, the techniques utilized and indications for this therapy. Prolotherapy is an extremely successful therapy in experienced hands and offers powerful pain relief and healing, especially to those who cannot afford PRP or Stem Cells and is therefore, a therapy that be part of all physicians training in third world countries and the like where large percentages of the population cannot afford or have no access to PRP or stem cells. For an indigent population, prolotherapy offers a very good treatment for healing and relief of musculoskeletal pain. Any physician doing stem cell therapy (musculoskeletal) should be well trained in prolotherapy prior to using the advanced therapies of performing platelet rich plasma injections (PRP) and bone marrow aspirates for stem cells and liposuctions for adipose derived stem cells. These procedures are very expensive as compared to prolotherapy. You need to have a major knowledge of injection techniques and know your anatomy-bones, ligaments, tendons and muscles in order to put the cells where they need to be. You need to be able to do these procedures via head, heart and hand (palpation) and via ultrasonic guidance and C-arm Fluoroscopy for more precision. Before rushing into doing stem cell therapies, prolotherapy should be tried because it is a much simpler and less invasive technique. Prolotherapy is currently being performed as a precursor to having PRP and stem cell therapy by competent regenerative medicine doctors. A discussion of what is prolotherapy and its uses and what it does is germane to this talk. It is important for physicians to know that often a simple treatment can be performed to accomplish the same result as long as one is experienced and knowledgeable as a prolotherapist.