The following student awards were presented at the 2011 In Vitro Biology Meeting, Raleigh, North Carolina. Information on additional awardees at the 2011 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 ( or by contacting the SIVB Business Office at (919) 562-0600,, or Dr. Pamela Weathers, Chair, Student Affairs and Awards Committee, at


Anna de La Fuente

Identification of Cadmium-regulated miRNAs in Rat Renal Proximal Tubule Epithelial NRK-52E Cells

MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional regulators of gene expression. Cadmium (Cd) is a major nephrotoxic environmental pollutant that selectively damages the proximal tubule. The objective of the present study was to examine the effect of Cd on miRNA expression in rat kidney proximal tubule epithelial NRK-52E cells to determine their potential involvement in Cd-induced nephrotoxicity. The NKR-52E cells were exposed to 0-10 µM CdCl2 in a serum-containing DMEM media for 24 hours. MicroRNAs were extracted using the mirVana microRNA isolation kit and analyzed using a rat miRNA LC Sciences microarray. The results showed that treatment of cells with 5 µM of CdCl2 causes phenotypic cellular alterations such as cell separation without causing overt cellular toxicity, as assessed by trypan blue and WST-1 assay. The results of the microRNA microarray analysis showed that acute exposure to CdCl2 significantly down-regulated rno-miR-30c and rno-miR-26a (t-test, p < 0.05), rno-miR-125a-5p and rno-miR-107 (p < 0.1), and significantly up-regulated rno-miR-466b-2, rno-miR-3584-5p, rno-miR-29a (p < 0.1). Nineteen additional miRNAs appeared to be altered following exposure to CdCl2 but low signal intensity precluded more detailed analysis. The present research indicates that exposure to non-cytotoxic concentrations of CdCl2 affects the expression of miRNAs in NRK-52E cells and raises the possibility that alteration in levels of miRNAs may play a role in Cd-induced proximal tubule injury.

Anna de La Fuente, Midwestern University, 555 31st St., Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 47:S31-32, 2011



Liwen Fei

From Cells to Field-ready Plants: One-step Micropropagation in a Mist Bioreactor

Micropropagation is a widely applied technique for rapid regeneration of whole plants in large numbers using plant tissues ranging from single cells to shoot segments. The scaling of this technique, on the other hand, is hampered by its high labor costs for subculture and acclimatization. A mist bioreactor may provide cost effective scale-up of the entire process of micropropagation beginning with cells to field-ready plants in one step. This bioreactor is mainly composed of a growth chamber made of disposable plastic, an ultrasonic nozzle with a conical tip, gas inlet, medium reservoir, timer and peristaltic pump. Using carrot as a model plant, undifferentiated carrot cells were sprayed through the ultrasonic nozzle onto nylon screens placed in the growth chamber, fed with B5 medium mist in different feeding cycles and harvested after 2 weeks to measure both cell viability, after this mode of inoculation, and development of embryos. After inoculation using 4.5 watts of ultrasonic power, 51.2% cells remained viable. Using a feeding cycle of 1min mist on every 15 min at 38ml/min, the total embryos produced per gram fresh weight were 11,322 and 10,138 for nylon screens with openings of 50 micron (CMN50) and 90 micron (CMN90), respectively. Of these embryos, there was no difference in which nylon screen was used; about 3.5% were developed to the torpedo or cotyledon stage. When a more frequent misting cycle was used that fed the same volume of medium per hour, there was no significant change in embryo formation. On the other hand, the yield of embryos beyond the heart stage tripled. This ongoing study includes additional experiments to maximize embryo formation with full development to field-ready plants and scale-up.

Liwen Fei, Worcester Polytechnic Institute, 100 Institute Road, Department of Biology and Biotechnology, Worcester, MA 01609. In Vitro Cellular and Developmental Biology, 47:S31-32, 2011



Vinita C. Daniel

Cullin-5 Knock-down Affects Gene Expression in Human Breast Cancer Cells

Breast cancer is the second leading cause of cancer-related death in women in the United States. The Cullin-5 (Cul5) gene is thought to play a role as a tumor suppressor in the development of breast cancer since breast cancer tissue commonly demonstrates decreased expression versus matched normal tissue. Cul5 is known to function in the ubiquitin-mediated protein degradation pathway; however, the tumor suppressor mechanisms are not well understood. To investigate the tumor suppressor roles of Cul5, an RNAi model of Cul5 knock-down using siRNAs and the MDA-MB-231 breast cancer cell line was used to screen for changes in gene expression using microarrays. A negative control or Cul5 siRNA were introduced into the cells using reverse transfection or electroporation, and knock-down was monitored using RT-PCR and Western blot analysis. Total RNA from the siRNA treated cells was used to screen for changes in gene expression using Human Breast Cancer and Estrogen Receptor, Human Tumor Metastasis, Human Cell Cycle and Human Apoptosis RT2 Profiler™ PCR Arrays. Cul5 knock-down was confirmed at the mRNA level in both electroporated and transfected cells as evidenced by the 80.4% and 58.7% decrease in Cul5 mRNA expression, respectively. Cul5 protein knock-down was also documented in siRNA transfected cells. Genes that were up or down regulated (≥1.5 fold) in a consistent manner with the two siRNA delivery methods were considered for future validation. Genes that were up regulated include MUC1 and KISS1. Genes that were down regulated include CIDEA, CD70, TNFSF8, LTA, CASP3, IL6, IL1B and CLU. The identification of genes that are differentially regulated in cancer cells with decreased expression of Cul5 may provide insight into the tumor suppressor functions of Cul5 in breast cancer.

Vinita C. Daniel, Midwestern University, 555 31st St., Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 47:S31, 2011.



Debora Araujo Esposito

Brassinosteroids Increase Protein Accumulation in Skeletal Muscle Cells

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5a-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids. We recently showed that a natural brassinosteroid (22S,23S)-homobrassinolide (HB) [1] stimulates protein synthesis, inhibits protein breakdown, and increases muscle mass in rats, but has very low androgenic activity both in vitro and in vivo. In this study we synthesized a set of brassinosteroid analogues including (22S,23S)-homocastasterone [2], (22S,23S)-3a-fluoro-homobrasinolide [3], (22S,23S)-3a-fluoro-homocastasterone [4], (22S,23S)-7v-aza-homobrassinolide [5], (22S,23S)- 6-aza-homobrassinolide [6], and studied their anabolic efficacy in L6 rat skeletal muscle cells in comparison to other synthetic and naturally occurring brassinosteroids (22R,23R)- homobrassinolide [7], (22S,23S)-epibrassinolide [8], and (22R,23R)-epibrassinolide [9]. Presence of the 6-keto group in the B ring and stereochemistry of 22a,23a-vicinal hydroxyl groups in the side chain were critical for the anabolic activity, possibly due to higher cytotoxicity of the 22b, 23b-hydroxylated brassinosteroids. All anabolic brassinosteroids tested in this study selectively activated PI3K/Akt signaling pathway as evident by increased Akt phosphorylation in vitro. The pharmacogenomic properties of HB were characterized in healthy rats orally administered with 60 mg/kg HB for 24 days and indicated that HB potently stimulated two sets of genes involved in muscle cell growth and carbohydrate metabolism. In summary, plant brassinosteroids and their synthetic derivatives may offer a novel therapeutic strategy for promoting growth, repair, and maintenance of skeletal muscles.

Debora Araujo Esposito, Graduate Research Assistant, Rutgers University, 59 Dudley Road, Foran Hall – Room 128, New Brunswick, 08901. In Vitro Cellular and Developmental Biology, 47:S38, 2011.



Ergun Kaya

In Vitro Propagation and Cryopreservation of an Endemic Species of Turkey, “T. cariensis Hub.-Mor. & Jalas.

Among the aromatic plants belonging to Lamiaceae family, the genus Thymus is remarkable for the numerous species and varieties of wild-growing plants, many of which are typical for the Mediterranean area. In Turkey, Thymus is represented by 38 species, with a 53%  of endemism ratio. Present study investigated in vitro propagation and cryopreservation of Thymus cariensis Hub.-Mor. & Jalas, an endemic species from western Anatolia. 1-1.5 cm long shoot tips, excised from mature plants of T. cariensis, in Mugla, were decontaminated and used to initiate in vitro shoot cultures Semi-solid MS medium, supplemented with 1 mg l-1 kinetin and 0.3 mg l-1 GA3, provided 95.7% of regeneration, with 1.27 shoots produced per explant. Shoot rooting was obtained on semi-solid MS medium, supplemented with 0.05 mg l-1 2,4-D. Indeed, this medium formulation enabled 100% rooting, each microshoot having 11.95 adventitious roots, on average. Rooted plantlets were transferred to 250-ml plastic pots and acclimatized successfully by gradually reducing the relative humidity. As for cryopreservation, following 2-week cold hardening (at 4°C, in darkness) of in vitro shoot cultures and 48-h sucrose preculture (on semi-solid MS medium, containing 0.25M sucrose) of excised shoot tips, vitrification-based one-step freezing approach, ‘droplet-freezing’, was applied. Accordingly, shoot tips were placed into 4-5 µl PVS2 drops on sterile aluminium foil strips (~ 5 x 15 mm) resting in a Petri dish on ice block (3 drops per aluminium foil), and treated with the solution for 15, 30, 45, 60, 75, 90, 105 or 120 min. Aluminium foils were then transferred into cryovials and directly plunged into liquid nitrogen (LN). Thawing was done at room temperature by removing the aluminium foils from LN and immersing them in washing solution (i.e., liquid MS medium, containing 1.2M sucrose). When the explants were totally melted, they were transferred on above-mentioned regeneration medium and incubated in standard culture conditions for recovery. Using this approach, 20% viability and 18.3% recovery of T. cariensis shoot tips was obtained following 90-min PVS2 treatment. To our knowledge, this is the first report where in vitro propagation and cryopreservation of T. cariensis is succeeded.

Ergun Kaya, Gebze Institute of Technology, Istanbul cad. no:101, Kocaeli, 41400 Turkey. In Vitro Cellular and Developmental Biology, 47:S63-64, 2011.



Sachiko Yamasaki

Long-term Serial Cultivation of Mouse Induced Pluripotent Stem (iPS) Cells in Serum-free and Feeder-free Defined Medium

Mouse Embryonic Stem (mES) cells and mouse induced Pluripotent Stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture media containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mouse ES cells can be maintained in undifferentiated state without feeder cells. Using this culture condition, the miPS cells are able to be maintained in ESF7 medium for more than 1 year with an undifferentiated phenotype by virtue of the expression of the transcription factor Oct-3/4, Nanog, Sox2, Esg1, and alkaline phosphatase, and the cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor (FGF)-2 with heparin induced miPS cells to differentiate into neuronal- and glial-like cells both in an adherent monolayer culture and in suspension embryoid body culture. miPS cells maintained both in ESF7 and in conventional serum-supplemented medium with feeder cells transplanted subcutaneously into the back skin of SCID mice, both produced tumors with tissues of all three germ layers. As this simple serum-free adherent monoculture system supports the long term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate the cell responses to growth factors under defined conditions, and could provide useful information to develop the feeder-free and serum-free medium of human iPS cells for clinical use.

Sachiko Yamasaki, Division of Frontier Medical Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi1-2-3 Minami-ku Hiroshima, Hiroshima City, 7348553, Japan. In Vitro Cellular and Developmental Biology, 47:S45, 2011.

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