Cell culture media and supplements In name of Allah Cell culture media and supplements Dr Shafaei

Contd.. 1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells. 1923: Carrel and Baker developed 'Carrel' or T-flask as the first specifically designed cell culture vessel. They employed microscopic evaluation of cells in culture. 1925:subculthre of firbroblastic cell line.

Contd.. 1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture. 1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium (DMEM). 1961: Hayflick and Moorhead isolated human fibroblasts (WI-38) and showed that they have a finite lifespan in culture. 1965: Ham introduced the first serum-free medium which was able to support the growth of some cells (Hams).

Contd.. 1975: Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal antibody. 1982: Human insulin became the first recombinant protein to be licensed as a therapeutic agent. 1985: Human growth hormone produced from recombinant bacteria was accepted for therapeutic use. 1986: Lymphoblastoid γIFN licensed. 1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available. 1989: Recombinant erythropoietin in trial. 1990: Recombinant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF, EGF, mAbs, IL-2).

Adult stem cells: A brief history Adult stem cell research began about 40 years ago Bone marrow stromal cell discoveries in 1960s Adipose derived stem cells are isolated in 2001.

Feeder layer

For Cross contamination elimination

Stem cell niches Niche: Microenvironment around stem cells that provides support and signals regulating self-renewal and differentiation stem cell niche Direct contact Soluble factors Intermediate cell Stem cell niches The stem cell niche is a major concept in stem cell biology. Understanding the microenvironment around stem cells is as important as understanding stem cells themselves. The microenvironment regulates the behavior of stem cells and thus can teach us how to control stem cells in culture. The niche can act on a stem cell by various mechanisms: Direct contact between the stem cell and the niche cells Soluble factors released by the niche that travel to the stem cell Intermediate cells that ‘communicate’ between the niche and the stem cell Scientists are still working to understand exactly how niches work, and more is known about the niches of some kinds of stem cells than others.

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.

A growth medium or culture medium is a liquid or gel designed to support the growth of cells

Types of Cell Culture Media 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 PBS, DPBS, HBSS, EBSS Basal media MEM DMEM Complex media RPMI-1640, IMDM

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

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

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 .

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

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)

Criteria for Selecting Media

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 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 COS-7 Monkey DMEM+ 2mM Glutamine + 10% FBS Gene expression and virus replication studies CHO Hamster Ham′s F12 + 2mM Glutamine + 10% FBS Nutritional and gene expression studies HEK 293 EMEM (EBSS) + 2mM Glutamine + 1% Non Essential Amino Acids (NEAA) + 10% FBS Transformation studies HUVEC Endothelial F-12 K + 10% FBS + 100 µg/ml Heparin Angiogenesis studies Jurkat RPMI-1640 + 10% FBS Signaling studies

Common media and their applications 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

Brain-derived neurotrophic factor

Media Supplements Serum in Media Antibiotics 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

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 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.

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

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 H2O2 which is directly toxic to cells

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