Animal cell culture media

different types of animal cell culture media

1. Animal cell culture media

2. APPLICATIONS OF TISSUE CULTURE Studies on intracellular activity eg . cell cycle and differentiation, metabolism Elucidation of intracellular flux eg . hormonal receptors, signal transduction Studies related to cell to cell interaction eg . cell adhesion and motility, metabolic cooperation Evaluation of environmental interactions eg . cytotoxicity, mutagenesis Studies dealing with genetics eg . Genetic analysis, immortalization, senescence Laboratory production of medical/ pharmaceutical compounds for wide range of applications eg . Vaccines, interferon, hormones 2

3. Animal cell culture are useful for the production of many pharmaceutically/ medically important proteins which are as follows: Plasminogen Interferon Blood clotting factors Hormones Monoclonal antibodies Erythropoietin 3

4. Major development’s in cell culture technology First development was the use of antibiotics which inhibits the growth of contaminants. Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel Third was the use of chemically defined culture medium. 4

5. Culture medium definition: A growth medium or culture medium is a liquid or gel designed to support the growth of cells 5

6. Media Type Examples Natural media Biological Fluids plasma, serum, lymph, human placental cord serum, amniotic fluid Tissue Extracts Extract of liver, spleen, tumors, leucocytes and bone marrow, extract of bovine embryo and chick embryo Clots coagulants or plasma clots Artificial media Balanced salt solutions Eagle’s BS , Hank’s BS, Basal media MEM DMEM Complex media RPMI-1640, IMDM 6 Types of Cell culture media

7. Natural Media Very useful Lack of knowledge of the exact composition of these natural media 7

8. Artificial Media Serum containing media Serum-free media (defined culture media) Chemically defined media Protein-free media 8

9. Basic Components of Culture Media Culture media ( as a powder or as a liquid) contains: amino acids Glucose Salts Vitamins Other nutrients The requirements for these components vary among cell lines, and these differences are partly responsible for the extensive number of medium formulations . 9

10. Natural buffering system HEPES Phenol red as a pH indicator (yellow or purple) Inorganic salt Amino Acids (L-glutamine) Carbohydrates Proteins and Peptides (important in serum-free media. Serum is a rich source of proteins and includes albumin, transferrin, aprotinin , fetuin , and fibronectin Fatty Acids and Lipids Vitamins Trace Elements 10

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12. Common Cell Culture Media Eagle’s Minimum Essential Medium (EMEM ) Dulbecco’s Modified Eagle’s Medium (DMEM ) Low glucose High glucose RPMI-1640 Ham’s Nutrient Mixtures DMEM/F12 Iscove’s Modified Dulbecco’s Medium (IMDM) 12

13. Criteria for Selecting Media 13

14. Cell Line Morphology Species Medium Applications HeLa B Epithelial Human MEM+ 2mM Glutamine+ 10% FBS + 1% Non Essential Amino Acids (NEAA) Tumourigenicity and virus studies HL60 Lymphoblast Human RPMI 1640 + 2mM Glutamine + 10-20% FBS Differentiation studies 3T3 clone A31 Fibroblast Mouse DMEM + 2mM Glutamine +5% New Born Calf Serum (NBCS) + 5% FBS Tumourigenicity and virus studies COS-7 Fibroblast Monkey DMEM+ 2mM Glutamine + 10% FBS Gene expression and virus replication studies CHO Epithelial Hamster Ham′s F12 + 2mM Glutamine + 10% FBS Nutritional and gene expression studies HEK 293 Epithelial Human EMEM (EBSS) + 2mM Glutamine + 1% Non Essential Amino Acids (NEAA) + 10% FBS Transformation studies HUVEC Endothelial Human F-12 K + 10% FBS + 100 µg/ml Heparin Angiogenesis studies Jurkat Lymphoblast Human RPMI-1640 + 10% FBS Signaling studies 14

15. Common media and their applications Media Tissue or cell line IMDM Bone marrow, hematopoietic progenitor cells, human lymphoblastoid leukemia cell lines MEM Chick embryofibroblast , CHO cells, embryonic nerve cells, alveolar type cells, endothelium, epidermis, fibroblast, glia, glioma , human tumors, melanoma DMEM Mesenchymal stem cell, chondrocyte, fibroblast, Endothelium , fetal alveolar epithelial type II cells, cervix epithelium, gastrointestinal cells, mouse neuroblastoma , porcine cells from thyroid glands, ovarian carcinoma cell lines, skeleton muscle cells, sertoli cells, Syrian hamster fibroblast RPMI-1640 T cells and thymocytes , hematopoietic stem cells, human tumors, human myeloid leukemia cell lines, human lymphoblastoid leukemia cell lines, mouse myeloma, mouse leukemia, mouse erythroleukemia , mouse hybridoma , rat liver cells Nutrient mixture F-10 and F-12 Chick embryo pigmented retina, bone, cartilage, adipose tissue, embryonic lung cells, skeletal muscle cells 15

16. Media Supplements Serum in Media Basic nutrients Growth factors and hormones Binding proteins Promote attachment of cells to the substrate Protease inhibitors Provides minerals, like Na+, K+, Zn2+, Fe2+, etc Protects cells from mechanical damages during agitation of suspension cultures Acts a buffer Antibiotics 16

17. Aseptic conditions Switch on the laminar flow cabinet 20 mts prior to start working Swab all bottle tops & necks with 70% ethanol If working on the bench use a Bunsen flame Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger Work either left to right or vice versa, so that all material goes to one side, once finished 17

18. Clean up spills immediately & always leave the work place neat & tidy Never use the same media bottle for different cell lines. If caps are dropped or bottles touched unconditionally touched, replace them with new ones Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C Never use stock of materials during handling of cells. 18

19. Contaminant’s of cell culture Cell culture contaminants of two types Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible Biological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines 19

20. Effects of Biological Contamination’s They competes for nutrients with host cells Secreted acidic or alkaline by-products ceases the growth of the host cells Degraded arginine & purine inhibits the synthesis of histone and nucleic acid They also produces H 2 O 2 which is directly toxic to cells 20

21. Detection of contaminants In general: turbid culture media, change in growth rates, abnormally high pH, poor attachment, multi-nucleated cells, graining cellular appearance, vacuolization, inclusion bodies and cell lysis Yeast, bacteria & fungi usually shows visible effect on the culture (changes in medium turbidity or pH) Mycoplasma detected by direct DNA staining with intercalating fluorescent substances e.g. Hoechst 33258 Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNA The best and the oldest way to eliminate contamination is to discard the infected cell lines directly 21

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