Culture Media

Report
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).
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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.
For Cross contamination elimination
Stem cell niches
Niche:
Microenvironment around stem cells that
provides support and signals regulating selfrenewal and differentiation
Direct contact
Soluble factors
stem cell
niche
Intermediate cell
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
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
Natural media
Balanced salt solutions PBS, DPBS, HBSS, EBSS
Artificial media
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
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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 .
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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
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RPMI-1640
Ham’s Nutrient Mixtures
DMEM/F12
Iscove’s Modified Dulbecco’s Medium (IMDM)
Criteria for Selecting Media
Cell Line
Morphology
Species
HeLa B
Epithelial
HL60
Lymphoblast Human
3T3 clone
A31
Fibroblast
COS-7
CHO
Fibroblast
Epithelial
Human
Medium
MEM+ 2mM Glutamine+ 10% FBS +
1% Non Essential Amino Acids
(NEAA)
RPMI 1640 + 2mM Glutamine + 1020% FBS
Applications
Tumourigenicity
and virus studies
Differentiation
studies
Mouse
DMEM + 2mM Glutamine +5% New Tumourigenicity
Born Calf Serum (NBCS) + 5% FBS
and virus studies
Monkey
Gene expression
and virus
DMEM+ 2mM Glutamine + 10% FBS
replication
studies
Hamster
Nutritional and
Ham′s F12 + 2mM Glutamine + 10%
gene expression
FBS
studies
EMEM (EBSS) + 2mM Glutamine +
1% Non Essential Amino Acids
(NEAA) + 10% FBS
Transformation
studies
F-12 K + 10% FBS + 100 µg/ml
Heparin
RPMI-1640 + 10% FBS
Angiogenesis
studies
Signaling studies
HEK 293
Epithelial
Human
HUVEC
Endothelial
Human
Jurkat
Lymphoblast Human
Common media and their applications
Media
IMDM
MEM
DMEM
RPMI-1640
Tissue or cell line
Bone marrow, hematopoietic progenitor cells, human
lymphoblastoid leukemia cell lines
Chick embryofibroblast, CHO cells, embryonic nerve cells,
alveolar type cells, endothelium, epidermis, fibroblast, glia,
glioma, human tumors, melanoma
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
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 Chick embryo pigmented retina, bone, cartilage, adipose tissue,
F-10 and F-12
embryonic lung cells, skeletal muscle cells
Media Supplements
• Serum in Media
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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
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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
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