Cell culture media and supplements

Cell culture media and supplements

In name of Allah

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.

For Cross contamination elimination

Stem cell niches

Direct contact Soluble factors Intermediate cell

stem cell

niche

Niche:Microenvironment around stem cells that provides support and signals regulating self-renewal and differentiation

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 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

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

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

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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