SAMBANIS,ATHANASSIOS

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Final Report on Cryopreservation of Biological Tissues

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SANDIA REPORTSAND2001-1639Unlimited ReleasePrinted June 2001Final Report on Cryopreservation ofBiological TissuesAthanassios SambanisPrepared bySandia National LaboratoriesAlbuquerque, New Mexico  87185 and Livermore, California  94550Sandia is a multiprogram laboratory operated by Sandia Corporation,a Lockheed Martin Company, for the United States Department ofEnergy under Contract DE-AC04-94AL85000.Approved for public release; further dissemination unlimited.Issued by Sandia National Laboratories, operated for the United States Departmentof Energy by Sandia Corporation.NOTICE:  This report was prepared as an account of work sponsored by an agencyof the United States Government.  Neither the United States Government, nor anyagency thereof, nor any of their employees, nor any of their contractors,subcontractors, or their employees, make any warranty, express or implied, orassume any legal liability or responsibility for the accuracy, completeness, orusefulness of any information, apparatus, product, or process disclosed, or representthat its use would not infringe privately owned rights. Reference herein to anyspecific commercial product, process, or service by trade name, trademark,manufacturer, or otherwise, does not necessarily constitute or imply its endorsement,recommendation, or favoring by the United States Government, any agency thereof,or any of their contractors or subcontractors.  The views and opinions expressedherein do not necessarily state or reflect those of the United States Government, anyagency thereof, or any of their contractors.Printed in the United States of America. This report has been reproduced directlyfrom the best available copy.Available to DOE and DOE contractors fromU.S. Department of EnergyOffice of Scientific and Technical InformationP.O. Box 62Oak Ridge, TN  37831Telephone: (865)576-8401Facsimile: (865)576-5728E-Mail: reports@adonis.osti.govOnline ordering:  http://www.doe.gov/bridgeAvailable to the public fromU.S. Department of CommerceNational Technical Information Service5285 Port Royal RdSpringfield, VA  22161Telephone: (800)553-6847Facsimile: (703)605-6900E-Mail: orders@ntis.fedworld.govOnline order:  http://www.ntis.gov/ordering.htmSAND2001-1639 Unlimited Release Printed June 2001 Final Report on Cryopreservation of Biological Tissues Athanassios Sambanis Georgia Institute of Technology Parker H. Petit Institute for Bioengineering and Bioscience 281 Ferst Drive, NB . Atlanta, Georgia 30332-0363 Abstract This report summarizes the experimental and anayltical work that was done in support of Sandia National Laboratories. This work investigated the feasibility of obtaining validation data to support the development of a complex computer code that simulates the thermal and chemical response of living tissue to a freezing transient. . 3 (this page intentionally left blank) This is to report on the work that we did on the contract from Sandia on the project entitled “Cryopreservation of Biological Tissues,” with the performance period May 24 2000, to Sep- tember 15 2000. Although the time was short, I believe we made significant progress towards addressing our research objectives and developing a long-term collaboration on modeling and experimental studies in the area of tissue engineering. The specific tasks of the contractor (Georgia Tech) and the work accomplished in each are as follows. 1. Determine the type of data which are (i) experimentally feasible and (ii) suitable for model validation. In collaboration with our technical contact at Sandia (Dr. Ronald Dykhuizen), we identified that the appropriate type of data to be collected include (i) the size of cells and (ii) the con- centration of cryoprotective agent (CPA) as a function of radial position in spherical encapsu- lated cell constructs and following freezing/thawing procedures. 2. Perform a literature search to identify similar experiments. We performed a thorough literature search, which yielded no publications with experiments of the same type. However, we did identify articles describing other useful studies, including the freezing of hepatocytes in different types of extracellular matrix. The following article discusses the cryopreservation of alginate-entrapped cells. It indicates that alginate doesn’t have many of the problems encountered by others when using collagen as an extracellular matrix.: Guyomard, Claire, Rialland, Laure, Fremond, Benjamin, Chesne, Christophe, Guillouzo, Andre, “Influence of Alginate Gel Entrapment and Cryopreservation on Survival and Xenobi- otic Metabolism Capacity of Rat Hepatocytes.” Toxicology and Applied Pharmacology, 14 1: 349-356 (1996) The following three articles discuss some of the problems caused by collagen: Cattral, Mark S., Lakey, Jonathan R.T., Wamock, Garth L., Kneteman, Norman M., Rajotte, Ray V., “Effect of Cryopreservation on the Survival and Function of Murine Islet Isografts and Allografts.” Cell Transplantation, Vol. 7 No. 4: 3373-379 (1998) 5 Pajot-Augy, Edith, Prost, Rene, Axelos, Monque A.V., “Cryosolvents Effects on Low Tem- perature Gel Structure of Denatured Collagen.” Cryobiology, 28: 335-346 (1991) Watts, P., Grant, M.H., “Cryopreservation of rat hepatocyte monolayer cultures.” Human & Experimental Toxicology, 15: 30-37 (1996) 3. Develop a protocol for experiments, most likely using as a model system cells entrapped in spherical alginate beads. To address this, we began by verifying that an appropriate model system would be cells entrapped in spherical alginate beads and by identifying the appropriate experimental tech- niques for collecting the data identified in paragraph 1 above. We confirmed that calcium alg- inate gels can withstand freezing down to -2OC, -7OC, or -196C (boiling point of liquid nitrogen), followed by thawing to room temperature, without collapsing. It should be noted that, although no structural changes of the gels could be observed microscopically when freezing occurred to -20C or -7OC, such changes were noticed when beads were frozen to - 196C and then thawed to room temperature. In no case, however, did the gels collapse. To measure cell size distribution in beads, we decided to use confocal microscopy. To mea- sure the distribution of CPA, we decided it would be best to section frozen beads with a cryo- tome, then thaw the sections and assay the CPA amount in each. An undergraduate research assistant (Mr. Sunny Singh) working on this project during the contract period became famil- iar with confocal microscopy, however, technical difficulties with our cryosectioning appara- tus prevented him from developing sectioning protocols for the system at hand. 4. Perform experiments in which beads will be immersed in a solution at low temperature containing a cryoprotective agent and, following freezing, data will be collected. We did not manage to complete these experiments during the contract performance period for the following reasons: (i) these experiments are much more involved than initially antici- pated; (ii) we had technical difficulties with the cryosectioning apparatus during the summer. Nonetheless, the undergraduate research assistant became familiar with the techniques needed for these experiments, including cell culturing, cell encapsulation, freezing/thawing processes, cell counting and viability measurement, and confocal microscopy, and estab- lished most of the protocols needed for the proposed experiments. We are committed to con- tinue this line of research, which we believe is of profound significance for ensuring the off- the-shelf availability of tissue engineered products. Thus, Mr. Singh and Dr. Sambanis will work on these experiments beyond the end of the Sandia contract period with support from the Georgia Tech/Emory Center for the Engineering of Living Tissues (GTEC). In addition, we plan to hire a graduate student using GTEC funds to work on this project. 6 5. Provide Sandia with experimental details that will allow simulation of the experiments at Sandia. Please see paragraph 4 above for this. These experiments are in progress and data will be provided to Sandia as the experiments are performed. 6. Collaborate with Sandia to identify improvements to the model that will allow better agree- ment between experiments and predictions. In spite of the experimental delays described in paragraphs 4 and 5 above, Drs. Dykhuizen and Sambanis developed a realistic model that incorporates a number of changes and improvements relative to initial model version. A paper describing this modeling effort was recently submitted for publication in Cryobiology. In summary, we believe that we accomplished a significant amount of work during the perfor- mance period of this contract, and that we have set the stage for a long-term research effort in the area of cryopreservation of engineered tissues and of modeling tissue engineered substi- tutes. E \’ - 7 / (this page intentionally left blank) 8 Distribution Athanassios Sambanis Georgia Institute of Technology Parker H. Petit Institute for Bioengineering and Bioscience 281 Ferst Drive, NB Atlanta, Georgia 30332-0363 Sandia: 1 0826 W. L. Herrnina, 9 113 1 0834 A. C. Ratzel, 9110 1 0835 S. N. Kempka, ?141 1 0836 E. S. Hertel, 9116 5 0836 R. C. Dykhuizen, 9116 1 0841 T. C. Bickel, 9100 1 9018 Central Technical Files, 8945 1 2 0899 Technical Library, 96 16 1 0612 Review & Approval Desk, 9612 For DOE/OSTI 1 1030 Laboratory & University Res, 0188 9 (this page intentionally left blank) 

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Final Report on Cryopreservation of Biological Tissues

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