CELL CULTURES PART 1. TERMINOLOGY

CELL CULTURE Tissue culture is the term used for “the process of growing cells artificially in the laboratory” Tissue culture involves both plant and animal cells There are several advantages and disadvantages of using cell cultures

ADVANTAGES Use of animals reduced Cells are homogenous and have same growth requirements In vitro models allow for control of the extracellular environment Monitoring various elements and secretions without interference from other biological molecules that occurs in vivo

DISADVANTAGES Loss of in vivo environment such as hormones, support structures and various other chemicals that the cells interact with in vivo The artificial conditions could cause cells to de-differentiate, which will cause them to behave differently and produce proteins other than it would in vivo

TISSUE CULTURE CLASSIFICATION Primary Culture: Cells taken directly from a tissue to a dish Secondary Culture Cells taken from a primary culture and passed or divided in vitro. These cells have a limited number of divisions or passages. After the limit, they will undergo apoptosis.

PREPARATION OF PRIMARY CELL CULTURE

CELL LINES Cell Line: Cells that undergone a mutation and won’t undergo apoptosis after a limited number of passages. They will grow indefinitely. Transformed cell line A cell line that has been transformed by a tumor inducing virus or chemical. It can cause tumors if injected into animal. Hybrid cell line (hybridoma) Two cell types fused together with characteristics of each

PART 2. UNDERSTANDING CELL BEHAVIOR CELL CULTURES PART 2. UNDERSTANDING CELL BEHAVIOR

CONFLUENCY How “covered” the growing surface appears Optimal confluency for moving cells to a new dish is 70-80%: Too low  cells will be in lag phase and won’t proliferate Too high  cells may undergo unfavorable changes and will be difficult to remove from plate.

CONTACT INHIBITION When cells contact each other, they cease their growth (Cells arrested in G0 phase) Transformed cells will continue to proliferate and pile upon each other

ANCHORAGE DEPENDENCE Cells that attach to surfaces in vivo require a surface to attach to in vitro. Other cells or specially treated plastic or other biologically active coatings Blood cells are primary exception. Transformed cells may not require attachment.

PASSAGE NUMBER The number of times the cells have been removed (or “split”) from the plate and re-plated. Always write this on your plate or flask as P#

PART 3. SOLUTIONS USED IN CELL CULTURE CELL CULTURES PART 3. SOLUTIONS USED IN CELL CULTURE

Phosphate Buffered Saline (Ca2+ Mg2+ Free) Used to wash/remove excess serum that inhibits the function of Trypsin-EDTA Calcium will also inhibit the function of Trypsin-EDTA Must be warmed in the water bath before use so cells are not shocked by cold liquid.

Trypsin EDTA An enzyme used to detach the cells from a culture dish Trypsin cleaves peptide bonds (LYS or ARG) in fibronectin of the extracellular matrix EDTA chelates calcium ions in the media that would normally inhibit trypsin Trypsin will self digest and become ineffective if left in water bath more than 20 minutes Trypsinizing cells too long will reduce cell viability

TRYPTAN BLUE An exclusion dye Living cells cannot take up the dye and will appear bright and retractile Dead cells with broken membranes will absorb the dye and appear blue Usually add 200 ml of trypan blue to 200 ml of cell suspension in eppendorf tube

BLEACH Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can Add enough to change media to clear: wait 5 minutes, rinse solution down sink throw away the dish/flask/plate in the trash can

CELL CULTURES PART 4. EQUIPMENTS

CO2 incubator maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.

Water bath To warm media, TRED and PBS before placing on cells Can harbor fungi and bacteria, spray all items with 70% ethanol before placing in the hood. Usually takes 10 -15 minutes for media to warm, 5-10 for TRED to thaw

Vacuum pump For permanent aspiration of liquids (media, PBS and TRED) Use unplugged glass pasteur pipets, throw into sharps box when done.

Inverted Phase Microscope A phase contrast microscope with objectives below the specimen A phase plate with an annulus will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast

Uses of Animal Tissue Culture Growing viruses - these require living host cells Making monoclonal antibodies, used for diagnosis and research Studying basic cell processes Genetic modification & analysis

Uses of Animal Tissue Culture II ‘Knockout’ technology - inactivating certain genes and tracing their effects Providing DNA for the Human Genome Project (and other species’ genome projects)

ANTIBODIES STRUCTURE CLASS

ANTIBODIES POLYCLONAL. MONOCLONAL. Derived from different B Lymphocytes cell lines Derived from a single B cell clone mAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificity Batch to Batch variation affecting Ab reactivity & titre NOT Powerful tools for clinical diagnostic tests Enable the development of secure immunoassay systems.

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY

Ab titre reached in Serum PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum Spleen removed (source of cells)

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 2: - Screening Of Mice For Antibody Production After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery) Titre High Titre too low 2 weeks BOOST (Pure antigen) BOOST (Pure antigen)

+ PRODUCTION OF MONOCLONAL ANTIBODY 8 - Azaguanine Myeloma Cells HYBRIDOMA TECHNOLOGY Step 3: - Preparation of Myeloma Cells + 8 - Azaguanine Myeloma Cells Immortal Tumor Of Lymphocytes Myeloma Cells HGPRT- High Viability & Rapid Growth

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells PEG FUSION MYELOMA CELLS SPLEEN CELLS Feeder Cells Growth Medium Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas HYBRIDOMA CELLS ELISA PLATE HAT Medium

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 4: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or Expansion A. Clone Each +ve Culture B. Test Each Supernatant for Antibodies C. Expand +ve Clones Tissue Culture Method Mouse Ascites Method

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY

EVOLUTION OF MONOCLONAL ANTIBODY 1. TRANSGENIC DNA SPLICING / GENE KNOCK OUT 2. LIBRARIES a.BACTERIOPHAGE b. mRNA c. Cell Surface

ENGINNERED ANTIBODIES

FDA APPROVED MONOCLONAL ANTIBODIES H 2003 Psoriasis Raptiva™ Genentech/Xoma C 1997 Non-Hodgkin’s Lymphoma Rituxan® BiogenIdec/Genentech/Roche M Bexxar® Corixa/GlaxoSmithKline 2004 Colorectal Cancer Erbitux ™ BMS/ImClone Systems Asthma Xolair® Novartis/Genentech/Tanox PD 2002 Rheumatoid Arthritis Humira™ Abbott/CAT 2001 Chronic Lymphocytic Leukemia Campath® Schering /ILEX Oncology 2000 Acute Myleoid Leukemia Mylotarg™ Wyeth 1998 Acute Transplant Rejection Simulect® Novartis Crohn’s, Rheumatoid Arthritis Remicade® J & J Breast Cancer Herceptin® Avastin ® Genentech/Roche Viral Respiratory Disease Synagis® MedImmune/Abbott Zenapax® PDLI Zevalin™ BiogenIdec 1994 Acute Cardiac Conditions ReoPro® J&J/Eli Lilly 1986 Organ Transplant Rejection Orthoclone-OKT® Ortho Biotech Antibody Type (2) Date of FDA Approval Indications Name of Product(1) Company Name

Applications of Monoclonal Antibodies Diagnostic Applications Biosensors & Microarrays Therapeutic Applications Transplant rejection Muronomab-CD3 Cardiovascular disease Abciximab Cancer Rituximab Infectious Diseases Palivizumab Inflammatory disease Infliximab Clinical Applications Purification of drugs, Imaging the target Future Applications Fight against Bioterrorism