The following student awards were presented at the 2009 In Vitro Biology Meeting, Charleston, South Carolina. Information on additional awardees at the 2009 Meeting will be presented in the next issue of the In Vitro Report.  Information related to the available specific student awards can be found on the SIVB website (www.sivb.org) or by contacting the SIVB Business Office at (919) 420-7940, sivb@sivb.org, or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at weathers@wpi.edu.

2009 WILTON R. EARLE AND STUDENT TRAVEL AWARD

Summaria Zamurrad

Analysis of Extracellular Matrix Synthesized by Human Bronchial Smooth Muscle Cells In Vitro

The Extracellular matrix (ECM) consists of an intricate meshwork that provides structure and organization to tissues. The ECM forms a major component of the extra-cellular environment and helps regulate cell migration, signaling, adhesion, proliferation and differentiation.  Cells are constantly remodeling their microenvironment by synthesizing de novo ECM components according to their cell/organ specific functions. There is a need to develop in vitro models that replicate this native extracellular environment so that the tissue degradation seen in inflammatory diseases and metastasis can be studied more thoroughly. Our lab has been long involved in searching for such an optimal and stable ECM equivalent for use in in vitro assays.  A human cell based model would provide a more physiologically relevant substrate for the study of protease inhibitors.  Here we describe the initial synthesis and analysis of an insoluble ECM synthesized in vitro by normal Human Bronchial Smooth muscle cells (HBSmC).  Cells were plated at passages five and seven, grown to confluence, and then cultured for one or two weeks in media supplemented with ascorbic acid. Examination by phase contrast microscopy showed that two weeks of post confluent culture yielded increased ECM deposition for cells plated at either passage.  Radiolabeled precursors were added to quantitatively label matrix components.  The ECM composition was analyzed by sequential digestion with heparinase I & III, elastin adsorbed trypsin, chondroitinase ABC, collagenase, human neutrophil elastase (HNE) and proteinase K.  Preliminary data shows significant amounts of proline to be incorporated into the collagenase sensitive material, and methionine/cysteine in HNE sensitive material.  Based upon this pattern of amino acid incorporation and specific enzyme sensitivity, these components are presumed to be collagen and elastin respectively.  This ECM has potential for further study of matrix biology and utility as a model for wound healing studies, drug discovery, toxicology, tissue engineering and reparative medicine.

S. Zamurrad, Departments of Pathology and Biochemistry. SUNY Stony Brook. Stony Brook. NY 11790. In Vitro Cellular and Developmental Biology, 45:S50, 2009


2009 CELLULAR TOXICOLOGY AWARD AND JOSEPH F. MORGAN AWARD

Bryan Sansom

Comparison of Fish Cell Line Responses to Chemicals and Process-Affected Waters from the Oil Sands of Alberta, Canada

The extraction of bitumen from the Athabasca Oil Sands of Alberta, produces large volumes of tailings and process-affected waters (OSPW) that are stored on the operators’ leases before they are reclaimed.  Reclamation of the fluid tailings components of the OSPW proposes to utilize end pit lakes (EPLs) where the OSPW is isolated by a water cap in which a lake system will develop.  In order to be deemed successful, this lake must be capable of supporting a fish population.  However, until the lake is commissioned and allowed to undergo natural bioremediation processes over time, it is difficult to predict how various fish populations will perform with chronic exposure to OSPW.  Aspects of toxicity to biota, including fish, will be part of the evaluation of this technique.  Through the use of established fish cell-lines, but more importantly new cell-lines developed from native fish to the oil sands region, an economical and non-lethal bioassay is being developed and validated to support reclamation planning. This tool will aid in designing the continuous monitoring programs which will be implemented in the full-scale EPLs.  Use of experimental ponds/lakes containing OSPW provides a range of water quality for testing the suitability of this in vitro cell-line approach.  Because of the scale and species selectivity, such testing could circumvent the need for transporting and testing using currently available fish bioassays.   The fish cell line approach could lead to a prediction of fish survival and success in a range of OSPW impacted aquatic systems without the need to perform fish lethality tests.  With the fish in vitro approach, predicting performance capability within aquatic reclamation habitats and impacts of OSPW (runoff, seepage, groundwaters) to fish habitats in receiving aquatic systems (local streams, Athabasca River) could be accomplished without the need to sacrifice of large numbers of test animals.  Towards this goal, the present work evaluates the sensitivity of fish cell line bioassays and end-points to detect toxicity of oils sands chemicals such as naphthenic acids (NAs), polycyclic aromatic hydrocarbons (PAHs), surfactants and salts that have been shown to be the main toxic components of OSPW. Additionally, direct testing of OSPW samples on fish cells, by mixing the test waters with media salts and adding directly to the cells in culture without prior extraction procedures, and an innovative triple-cell-line assay for increased sensitivity of chemical exposures will be presented.

Bryan Sansom, University of Waterloo, Department of Biology and Wilfrid Laurier University, Waterloo, ON, CANADA.  In Vitro Cellular and Developmental Biology, 45:S45-46, 2009


2009 JOHN S. SONG AWARD AND STUDENT TRAVEL AWARD

Emrah Kirdok

Development of Procedures For Medium- and Long-term Storage of Coast Redwood (Sequoia sempervirens)

Sequoia sempervirens is a conifer tree, belonging to the genus Sequoia. The lumber, valued for light weight and resistance to decay, makes the tree one of the most valuable timber species. However, urbanisation, fires and storms give rise to the loss of this valuable germplasm. Today, biotechnological approaches offer a powerful alternative for safe storage of such genetic resources. Among these, ‘slow growth storage’ enables the storage of plant germplasm from several months to several years (medium-term storage) by reducing the growth rate of in vitro cultures in modified conditions. Cryopreservation, i.e. storage of plant germplasm at ultra-low temperatures (e.g. in liquid nitrogen at -196°C), where the biological reactions in the cells are hampered, makes available the storage of plant material for theoretically unlimited periods of time (long-term storage). Present study investigated the possibility of applying slow growth storage and cryopreservation to S. sempervirens. Former was achieved by maintaining encapsulated buds (in 3% Na-alginate solution) and in vitro shoot cultures at 4ºC, in darkness on four different media compositions (MS + 1mg/l BA, MS 0, QL + 1mg/l BA, QL 0) for 3-15 months, while latter was performed by pre-culturing the buds on sucrose-rich medium (0.12, 0.25 or 0.5 M for 24, 48 or 72 h), osmoprotection of the samples by PVS2 (15-120 min at 0°C) on aluminum foil strips (droplet freezing method) and direct immersion in liquid nitrogen. After at least 1 h of storage in liquid nitrogen, frozen samples were thawed by incubating them rapidly in 1.2 M sucrose solution and transferred to the recovery medium (QL + 20 gr/l activated charcoal). Outcomings of the study revealed the possibility of conserving in vitro shoot cultures at 4ºC and in darkness for a minimum of nine months without losing growth potential. As regards cryopreservation, optimizing pre-culture conditions previous to storage in liquid nitrogen and using droplet freezing approach for the induction of osmoprotection led to a satisfactory long-term storage of the buds with high post-thaw recovery rates.

Emrah Kirdok, Gebze Institute of Technology, Department of Biology, Istanbul cad., no: 101, 41400 Gebze, (Kocaeli), TURKEY. In Vitro Cellular and Developmental Biology, 45:S65-66, 2009

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