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Yakhnenko I.,Cryocore of the Stem Cell Center And | Yakhnenko I.,Celltronix | Wong W.K.,Sanford Burnham Institute for Medical Research | Katkov I.I.,Cryocore of the Stem Cell Center And | And 3 more authors.
Cryo-Letters | Year: 2012

Encapsulating insulin producing cells (INPCs) in an immunoisolation device have been shown to cure diabetes in rodents without the need for immunosuppression.!! However, microencapsulation in semi-solid gels raises longevity and safety concerns for future use of stem cell derived INPCs. We have focused on a durable and retrievable macro-encapsulation (>106 cells) device (TheraCyte™). Cryopreservation (CP) of cells preloaded into the device is highly desirable but may require prolonged exposure to cryoprotectants during loading and post-thaw manipulations. Here, we are reporting survival and function of a human islet cell line frozen as single cells or as islet-like cell clusters. The non-clusterized cells exhibited high cryosurvival after prolonged pre-freeze or post-thaw exposure to 10%-DMSO. However, both clusterization and especially loading INPCs into the device reduced viable yield even without CP. The survived cryopreserved macro-encapsulated INPCs remained fully functional suggesting that CP of macro-encapsulated cells is a promising tool for cell based therapies.

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.
Andrologia | Year: 2012

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.

PubMed | Celltronix, University of California at San Diego, Belgorod State University and Sanford Burnham Institute for Medical Research
Type: Journal Article | Journal: Cryobiology | Year: 2015

We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional slow freezing methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS+0.5M sucrose at 37C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions.

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 | Year: 2011

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.

Bolyukh V.F.,Celltronix | Oleksenko S.V.,Kharkiv Polytechnic Institute | Katkov I.I.,Celltronix
Refrigeration Science and Technology | Year: 2014

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.

Bolyukh V.F.,Kharkov National l Technology U. KhPI | Bolyukh V.F.,Celltronix | Katkov I.I.,Celltronix
ASME International Mechanical Engineering Congress and Exposition, Proceedings (IMECE) | Year: 2013

Main electromagnetic and thermal characteristics of cryoresistive windings (CRW) are systematized; and based on these results a mathematical model of an induction-dynamic device (IDD) is developed. Active elements of the IDD are cooled by liquid nitrogen. To calculate the IDD's working processes a numerical-analytical approach is used. Within a small interval of time analytical expressions for the main values are calculated. The transient process is modeled on a computer program by iterative relations and time loops. In the calculation the IDD's armature is represented as a set of elementary shortcircuited circuits uniformly distributed on a disk's surface, and the multi-turn inductor is represented by a primary circuit connected to the capacitance energy storage (CES). On the base of the carried out mathematical modeling it is demonstrated that the most effective is the cryogenic cooling of the armature for which a minimal amount of cryogen is required. At the simultaneous cooling of the inductor and the armature the IDD's efficiency increases 24 times. It is shown that in case of the use of a ferromagnetic core as an alternative to a cryogenic cooling, the IDD's efficiency increase is much lower. The validity of the proposed mathematical model and obtained numerical results is demonstrated on the base of experimental modeling. Copyright © 2013 by ASME.

Katkov I.I.,Celltronix | Bolyukh V.F.,Celltronix | Yakhnenko I.,Celltronix
ASME International Mechanical Engineering Congress and Exposition, Proceedings (IMECE) | Year: 2013

The levitation ('hovering') of a liquid droplet on the surface of the coolant such as liquid nitrogen (LN2) is a useful model for studying the Leidenfrost effecr (LFE), that is formation of a vapor film of boiling coolant around the surface of a relative,ly hotter sample, at cryogenic temperatures. Several models of the cryogenic droplet levitation (CDL) have been proposed but no experimental verifications had been proposed for this model in earlier papers. Utkan Demirci's group has recently developed fast ice-free cooling (vitrification) of microdroplets formed by an ink-jet printer. The group proposed a combination of a theoretical model of film boiling on a hot sphere with the zone theory of non-isothermal kinetic ice propagation within an initially liquid levitating droplet, and they gave theoretical predictions and experimental evaluations of the CDL (Leidenfrost) time tLF of droplets hovering on the surface of LN2 [6]. Here, we report our own experiment results of verification of the data and predictions reported in [6] and describe a thermodynamical model that for elucidating the fate of the levitating droplets. This model adequately explains our experimental results on measuring tLF but almost predicts somewhat 4-fold departure from the numbers claimed by Demirci's group. We also discuss possible flaws of the model and, especially, experimental claims presented in [6] © 2013 by ASME.

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.

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.

Embodiments of a cryopreservation device and method of use are described that allow cryopreserved samples to be transferred from one location to another. In one embodiment, a cryopreservation device comprises two, telescoping tubes; one for plunging biological material into a reservoir of cryogenic liquid refrigerant, and the other for capturing the biological material along with some of the liquid refrigerant, for transferring the biological material to another location.

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