Applications of Cell Culture in Biotechnology and Medicine (BCHB-536)

(Spring 2008; 2 credits)

Lecture: Thurs 4:00 pm to 6:00 pm Rm 341 (Jan. 17 - April 3, 2007) Lab: Wednesday 1 - 5 pm Rm LD4 (Jan. 30, Feb. 6, Feb. 13, Feb. 20) Course Director: Dr. Cynthia Rosenthal Course Description:
This course provides biochemistry, biomedical, and biotechnology graduate students with a basic understanding and hands-on experience on modern applications of tissue/cell culture technology in the study of diverse topics in life sciences, including molecular biology, toxicology, immunology and pharmacology. All GUMC graduate students are eligible to enroll in this course,with permission from the course director. Email: simbulac@georgetown.edu

Applications of Cell Culture in Biotechnology and Medicine (BCHB-536)

Course Objectives: The goal of Cell Culture Applications in
Biotechnology and Medicine is for students to acquire the necessary practical skills for the isolation of mammalian cells for in vitro studies, from frozen stock to initiation of cell cultures, maintenance of these cells in vitro, manipulation of cells in vitro, and application of biochemical/molecular techniques to in vitro situations. Due to time limitations, the course will focus on mammalian cell culture, although the same basic aseptic techniques can be used for the culture of insect, sh, plant or other cells. Lectures provide the background to understand the basic principles of cell culture.

Upon completion of the course, students will:

1. Develop basic aseptic skills for mammalian cell culture. 2. Develop prociency in establishment of primary cell culture and the maintenance of primary and immortalized cell lines. 3. Apply basic and specialized biochemical/ molecular techniques to in vitro studies. The laboratory practicum is a combination of laboratory exercises and lectures and includes the following: growth of adherent and nonadherent/suspension cells in vitro, routine maintenance, methods of cell enumeration and viability determinations, primary culture, cell cryopreservation, characterization, gene transfer/ cationic lipidmediated transient and stable transfection, cloning strategies (dilution cloning cloning), cell cycle analysis, cell fusion, cytotoxicity, and apoptosis measurements.

Applications of Cell Culture in Biotechnology and Medicine (BCHB-536)

Attendance Policy: You will be working in groups of three for the
duration of the semester. It is your responsibility to read the laboratory manual before and to attend each session. Failure to do so will place an undue burden on your lab partner(s). Attendance at ALL 4 laboratory sessions is mandatory. Any unexcused absence from lab will result in failure of the course. Lecture attendance is also required and will be reected in your participation grade.

Grading: There will be one mid-course exam (after lecture 4) and one
nal exam (after lecture 8). A portion of the nal grade will be based upon maintaining an up-to-date bound page-numbered notebook which will be provided at the start of the laboratory sessions. The students will record the objectives of each laboratory exercise, the detailed procedures performed in each laboratory exercise, data collected/ observations, and the discussion/ problems encountered, and conclusions drawn from each laboratory exercise in the notebook. Notebooks will be evaluated at the end of the course. The criteria for notebook evaluation will be maintaining complete and organized records for each exercise. The laboratory grade will also be partially evaluated on the basis of successful maintenance of uncontaminated cell lines, establishment of a primary culture, and derivation of clones by the end of the course. Exam I Exam II Class participation Laboratory notebook Total 30% 30% 10% 30% 100%

Applications of Cell Culture in Biotechnology and Medicine (BCHB-536) Spring 2008 (2 credits)
Lecture: Lecture 1 Introduction; biology of cultured cells; basic techniques Lecture 2 Primary culture; cell lines; cloning; selection; cell separation;characterization Lecture 3 Differentiation; transformation; quantitation Lecture 4 Cell proliferation; cell viability/toxicity; specialized cells Exam I Lecture 5 Tumor cells; organotypic culture; scale-up; molecular techniques Lecture 6 Flow cytometry; basics; cell cycle analysis; immunofluorescence; viability; apoptosis Lecture 7 Human stem cells: isolation, culture, characterization, differentiation of ES cells; Applications: ES Cell Research and ES Therapeutics Lecture 8 Molecular cytogenetic techniques: FISH; CGH, SKY Exam II Laboratory Lab 1 Basic procedures; cell lines - initiation of cell cultures; cell quantitation; cell viability; characterization-cell morphology; cell cycle analysis -ethanol fixation for DNA analysis Lab 2 Subculture; cryopreservation (cell freezing); primary culture - explant culture from tissue; characterization: immunocytochemical staining (Part I); gene transfer (Part I): transient transfection of cells using GFP-vectors; fluorescence microscopy: determination of transfection efficiency Lab 3 Cryopreservation (cell thawing); primary culture -transfer of explants and subculture; cell lines -maintenance of cell cultures; passaging; cloning -clonogenic assay (Part I): dilution cloning; immunocytochemical staining (Part II) Lab 4 Clonogenic assay (PartII): analysis; plating efficiency; isolation of clones; cell cycle analysis -flow cytometry; analysis of DNA histograms; apoptosis/necrosis assays: annexin V-PI staining; FACS analysis Submission of Lab notebooks: Feb. 29, 2008