• Core Objectives
Director: K. Pfenninger, MD (cell culture and biochemical analysis of the brain)
Co-Director: J. Sladek, PhD (neuroanatomy and histology)
The objective of this core is to stimulate and facilitate IDD-related research at the cellular and tissue levels.
The Cell Systems Core has two sections, focused on cell culture and on brain tissue, respectively. This core is designed to support in-vitro studies, including the use of human induced pluripotent stem cells(iPS cells), and to provide the technology and assistance to analyze brain tissue with neuroanatomical and biochemical techniques. The core also performs lymphoblast immortalization for bio-banking and genomic analysis.
Curt Freed, MD (iPS cells)
Renata Collard, PhD (cell culture)
Barbara Blanchard, MS (histology)
Insun Park, MS (cell culture)
Wenbo Zhou, PhD (iPS cells)
Investigators: Please remember to acknowledge your Colorado IDDRC affiliation and your use of the cores in all your published papers. For an example see the For Investigators web page.
Cell Culture Services:
Contact Person: Karl Pfenninger (firstname.lastname@example.org)
VIDEO: Cell Systems and Analysis core
1. In-vitro System Consulting.
K. Pfenninger, C. Freed and W. Zhou are available to IDDRC investigators for the evaluation of experimental approaches involving in-vitro systems. Options include the cultures of primary neurons or glial cells from wild-type or genetically engineered rodents, neurally differentiated iPS cells, and/or cell lines. Also part of the service is advice on the feasibility of visualizing particular functions or functional changes in cells using optical methods or biochemical approaches.
2. Neurally Differentiated Human iPS Cells.
Induced pluripotent stem cells or iPS cells can be generated from human fibroblasts. Such fibroblasts may originate from normal individuals or patients with a genetic disorder. The iPS cells can be differentiated into neurons and glia. The resulting cell cultures offer the opportunity to study a variety of functional parameters of normal and genetically deficient human neurons in vitro. This is a novel experimental system with very high potential for studying IDD pathogenesis mechanisms. The Cell Systems Core offers iPS cell generation and neural differentiation to IDDRC investigators. For further information, please contact W. Zhou or C. Freed.
3. Cell Lines and Primary Neural Cells.
The Core is a repository for a variety of cell lines and fibroblasts for IDD-related research use. Please inquire with R. Collard. In addition, the core can grow newly acquired cell lines for investigators and bank them for later use. In collaboration with the IDDRC’s Molecular Discovery Core, the Cell Systems and Analysis Core DNA-profiles human cell lines as needed to authenticate their identity. For investigators who wish to have many cell lines grown in large quantity, the core can provide this service if the work load permits, but the grant supporting the investigator’s study must buy personnel time in the core to perform this service. For pilot studies or small projects the Core provides cells (as available) at no charge.
The Core also provides training and assistance with the preparation of primary neural cell cultures. For experimentation with very limited scope or pilot studies, the Core prepares cultures of rat or mouse cortical explants or dissociated cells. For larger studies the principal investigator’s laboratory receives appropriate training and advice. Training also may include the establishment of other neuron populations (those from hippocampus, cerebellum, olfactory bulb, peripheral ganglia and others) or of glial cell types.
4. Lymphoblast Immortalization.
Immortalized lymphoblasts provide DNA in essentially unlimited quantities and enable analysis of chromosomes, copy number variations, RNA, etc. R. Collard and I. Park perform lymphoblast immortalization using EBV for the Translational Nexus.
5. Other Cell Culture Services.
a) Mycoplasma Testing: Upon request the cell culture core tests cells for potential mycoplasma contamination and provides this service for cultures grown and maintained in the different IDDRC investigator laboratories.
b) Cell Transfection: The Cell Culture Core has an Amaxa Nucleofector electroporator, which is the best currently available instrument for introducing vectors into difficult-to-transfect cell types, including primary neurons. This equipment, and advice on how to use it, are available to IDDRC members.
c) Cell Processing and Analysis: K. Pfenninger (with members of his laboratory) and R. Collard provide advice and training on how to handle cultures for live-cell imaging and how to process them for, e.g., immunolabeling or a variety of biochemical approaches.
Brain Tissue Services
Contact Person: John Sladek (email@example.com) or Karl Pfenninger (firstname.lastname@example.org)
1. Subcellular fractionation and biochemical analysis.
K. Pfenninger provides advice for the preparation and analysis of subcellular fractions such as synaptosomes from adult rodent brain, growth cones from fetal rodent brain, myelin, mitochondria, and other membrane fractions. Tools and instrumentation to perform such analyses are readily available in the IDDRC (see Molecular Discovery Core).
2. Neuroanatomy and Histology.
J. Sladek is available to advise investigators on the optimal neuroanatomical and -histological techniques to be used for examining the brains of experimental animals and on the interpretation of results. B. Blanchard is in charge of the various histology services.
VIDEO: Neuroanatomy and Histology services
a) Stereotaxic implantation of cells, factors and viral vectors. The Sladek laboratory has a rodent stereotaxic apparatus with a digital positioning manipulator that allows for a high degree of precision and reproducibility.
b) Brain perfusions can be performed on a wide variety of animal models that include rats, mice, guinea pigs, rabbits, and non-human primates with a range of routine to specialized fixatives. B. Blanchard is familiar with the advantages and disadvantages associated with a particular fixative or fixation method and can advise the members of the IDDRC of best uses and potential obstacles depending on their experimental needs. Expertise also is available in the perfusion of embryonic and postnatal animals, which requires special technical skill and knowledge of the appropriate fixation methods.
c) Neurohistochemical Preparation: B. Blanchard prepares tissue sections ranging in thickness from less than 5μ to 100μm or more, with the use of different embedding techniques, and with a rotary microtome, freezing sliding-blade microtome, cryostat, or Vibratome (for fresh tissue).
d) Staining: Routine histological preparation of tissue sections can be done with a variety of staining and counterstaining approaches, with dyes such as cresyl violet, methyl green, hematoxylin, and many others depending on the structure(s) needing to be visualized.
e) Immunohistochemical staining for various neuronal and glial markers, synaptic markers, indicators of differentiation or development, receptors, transmitters, enzymes and markers of inflammation. This may involve the application of double-labeling techniques, with the use of permanent dyes and chromagens, such as diaminobenzidine, nickel-enhanced diaminobenzidine, Vector Red, Vector Blue, and others, or fluorescent conjugated markers, such as fluorescein, rhodamine, Cy2, Cy3, Cy5, and AlexaFluors. The principal investigator’s laboratory provides the appropriate antibodies.
Lymphoblast Immortalization: These fees are included in the Translational Nexus Core.
iPS cell generalization and differentiation:
Cost per new cell line (usually 3 sublines): $3,500
Cost per existing control cell line: $1,500
For other pricing, please inquire.