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SAN DIEGO, CA, United States

In the companion paper, we discussed in details proper linearization, calculation of the inactive osmotic volume, and analysis of the results on the Boyle-vant' Hoff plots. In this Letter, we briefly address some common errors and misconceptions in osmotic modeling and propose some approaches, namely: (1) inapplicability of the Kedem-Katchalsky formalism model in regards to the cryobiophysical reality, (2) calculation of the membrane hydraulic conductivity Lp in the presence of permeable solutes, (3) proper linearization of the Arrhenius plots for the solute membrane permeability, (4) erroneous use of the term " toxicity" for the cryoprotective agents, and (5) advantages of the relativistic permeability approach (RP) developed by us vs. traditional (" classic") 2-parameter model. © 2011 Elsevier Inc. Source

Katkov I.I.,Celltronix | Katkov I.I.,Sanford Burnham Institute for Medical Research | Kan N.G.,Sanford Burnham Institute for Medical Research | Cimadamore F.,Sanford Burnham Institute for Medical Research | And 3 more authors.
Stem Cells International

Three modes for cryopreservation (CP) of human iPSC cells have been compared: STD: standard CP of small clumps with 10 of CPA in cryovials, ACC: dissociation of the cells with Accutase and freezing in cryovials, and PLT: programmed freezing of adherent cells in plastic multiwell dishes in a programmable freezer using one- and multistep cooling protocols. Four CPAs were tesetd: dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY). The cells in ACC and PLT were frozen and recovered after thawing in the presence of a ROCK inhibitor Y-27632 (RI). EG was less toxic w/o CP cryopreservation than DMSO and allowed much better maintenance of pluripotency after CP than PG or GLY. The cells were cryopreserved very efficiently as adherent cultures (+RI) in plates (5-6-fold higher than STD) using EG and a 6-step freezing protocol. Recovery under these conditions is comparable or even higher than ACC+RI. Conclusions. Maintenance of cell-substratum adherence is a favorable environment that mitigates freezing and thawing stresses (ComfortFreeze() concept developed by CELLTRONIX). CP of cells directly in plates in ready-to-go after thawing format for HT/HC screening can be beneficial in many SC-related scientific and commercial applications such as drug discovery and toxicity tests. Copyright © 2011 Igor I. Katkov et al. Source

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 140.00K | Year: 2012

DESCRIPTION (provided by applicant): Cryopreservation of germplasm (GP) (sperm, oocytes, embryos, stem cells, ovarian tissues) is essential for preserving the genetic variety of model animals, reproductive health in humans, the animal breeding industry andwildlife conservation. Although many methods, devices, and equipment exist both for slow freezing and fast cooling (vitrification), each method, cell type and species practically needs its own optimal preservation protocol. Vitrification (VF) is gaining in popularity with successful protocols being developed for many types of GP, including spermatozoa and stem cells. However, all existing VF methods require complicated and careful timing, may be prone to technical errors, often are not scalable, and are limited to very small sample volumes (0.5-5 ?L). As such, cryopreservation of samples such as semen, cord blood stem cells, and sufficient amounts of pluripotent stem cells and ovarian tissue is extremely difficult. The other aspect is that while th amount of potentially toxic cryoprotective agents (CPA) has been greatly reduced, the concentrations are still relatively high for the majority of GP types, and beside toxicity, the CPA addition and elution times must be precisely controlled. One of the major factors for vitrification is the critical cooling rate necessary for vitrification (Bcr), which strongly and inversely depend on the CPA concentration, For example, hundreds of thousands of ?C/min are needed to vitrify a water-glycerol solution that is tolerable for ALL CPA species concentrations. All existing methods purport to achieve such high speeds, but many have not in fact done so, mainly due to the Leidenfrost effect (LFE) - where a boiling nitrogen vapor coat forms around the sample. This vapor coatimpairs thermal conductivity by orders of magnitude and makes even droplets that are a fraction of a ? m L impossible to vitrify. With a speed around 500,000 ?K/min, we hypothesize that we can vitrify practically ALL species of germplasm using a unified method, equipment and supplies. Our Celltronix team has developed a completely new system for hyperfast cooling, called KrioBlast(r) , which completely eliminates LFE and can cool much larger samples than those currently used at rates of hundreds of thousands ?C/min. We have built a pilot model (first generation) of the system, the manually operated Krioblast-1, with which we could vitrify large sample volumes with dilute CPA solutions and also achieved some promising results for two trials on human and bull sperm. Upon obtaining a higher cooling rate, we will be close to devising a Universal Cryopreservation Protocol . In this Project, we will buil a semi-automatic system Krioblast-2, which would produce 2-3 fold faster cooling rates with a target of 200,000 ?C/min and vitrify cell volumes of up to 4,000 mL (1-2 orders of magnitude higher than is currently possible). We believe that such rates will be sufficient to vitrify all tyes of GP using a practically unified protocol. In Phase II, we will build aclosed modular stem for hyperfast cooling, cryogenic storage and shipment, and hyperfast thawing of cells and test Krioblast-3 on real germplasm cells. PUBLIC HEALTH RELEVANCE: Cryopreservation of germplasm (sperm, oocytes, embryos, stem cells, ovarian tissues) is essential for preserving the genetic variety of model animals, assisting human fertility techniques, the animal breeding industry, and wildlife conservation. A large variety of cryopreservation methods, devices and equipment currently exists, but each method, cell type and species would need its own optimal protocol. The goal of this Project is to develop a novel scalable device for hyper-fast (hundreds of thousands of ?C/min) cooling that would allow vitrification of a wide variety of germplasm cells and species using unified equipment and protocols, which will not only significantly benefit germplasm cryopreservation, but may eventually shift cryopreservation paradigms.

Merino O.,University of the Frontier | Sanchez R.,University of the Frontier | Risopatron J.,University of the Frontier | Isachenko E.,University of the Frontier | And 7 more authors.

The aims of this investigation were to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryo-injuries. Spermatozoa were isolated and vitrified using three different media: Group 1: standard buffer for fish spermatozoa, Cortland ® medium (CM, control); Group 2: CM+1% BSA+40% seminal plasma; and Group 3: CM+1% BSA+40% seminal plasma+0.125m sucrose. For cooling, 20-μl suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM+1% BSA at 37°C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility and cytoplasmic membrane integrity with SYBR-14/propidium iodide staining technique. Motility (86%, 81% and 82% for groups 1, 2 and 3, respectively) (P>0.1) was not decreased significantly. At the same time, cytoplasmic membrane integrity of spermatozoa of Groups 1, 2 and 3 was changed significantly (30%, 87% and 76% respectively) (P<0.05). All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility. However, cytoplasmic membrane integrity was maximal in Group 2 (CM+1% BSA+40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA+40% seminal plasma. © 2011 Blackwell Verlag GmbH. Source

Bolyukh V.F.,Celltronix | Oleksenko S.V.,Kharkiv Polytechnic Institute | Katkov I.I.,Celltronix
Refrigeration Science and Technology

The effects of cryogenic cooling of active elements by liquid nitrogen and the use of a ferromagnetic core on the efficiency of the linear induction-dynamic converter are considered. A mathematical model uses a set of coupled electromagnetic, thermal and mechanical equations. The experimental studies has confirmed the basic theoretical concepts and computational data that cryogenic cooling permits to increase the converter efficiency greater than in case of the use a ferromagnetic core alone. However, the high efficiency can be provided by only if cryogenic cooling of the armature is explored. Copyright © 2014 IIR/IIF. Source

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