Fig. 1: Steps of animal cell culture
(a) Requirements: Vertical Laminar air flow,
Incubator, Refrigerator, Microscope, Tissue culture ware.
(b) Temperature: The temperature is set at the same as the
body temperature of the host from which cells are procured. Most animal cells
require 36- 37 °C.
(c) Substrate: A good compatible substrate is
required for attachment and optimum growth. Glass and specially treated
plastics are commonly used as substrates. Thereafter, attachment factors such as
collagen, gelatin, laminin, etc., are used as substrate coating to improve the growth
and function of normal cells derived from the brain, blood vessels, kidney,
liver, skin, etc.
(d) Culture medium: It is an important and complex
factor for cell growth. The culture medium is supplemented with various growth
factors, pH, and osmolality regulators, and provides essential gases like oxygen
and carbon dioxide. The medium is also supplemented with various nutrients like
amino acids, vitamins, minerals, and carbohydrates, which are essential for the growth
of cells and provide energy for metabolism. The choice of media is based
on the cells being cultured. Generally, media like Dulbecco's modified Eagle's
medium (DMEM), Eagle's minimal essential medium (EMEM), and Glasgow Minimum
Essential Medium (GMEM) are used for cell culture. Prepared media is filtered
and incubated at 4 °C.
(e) Media and growth requirement: Temperature should be
maintained at 37°C, and the optimum pH is 7.2 to 7.5. The humidity is required
to be maintained properly in the media with a proper gas phase ratio
(Bicarbonate concentration and carbon dioxide in equilibrium). For the growth
of cultured cells, light intensity also plays a vital role. Inside the environment, cells are cultured in the dark because of light-induced production of toxic
compounds. Commonly used antibiotics are penicillin, streptomycin, Kanamycin,
etc. Commonly used fungicides are Nystatin and Amphotericin B. Bulk ions like
sodium, potassium, calcium, magnesium, chlorine, phosphorus, carbon dioxide,
oxygen, etc. Trace elements like iron, zinc, selenium, sugar, amino acids,
vitamins, choline, inositol, etc.
(f) Selection of organ: Different types of cells are
grown in culture, including connective tissue elements such as fibroblasts,
skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, and kidney),
and many different types of tumor cells. Based on morphology (shape and
appearance) or the functional characteristics of cells, they are divided into
three types.
• Epithelial-like - attached to a substrate and appears
flattened and polygonal in shape.
• Lymphoblast-like - cells do not attach and remain in
suspension with a spherical shape.
• Fibroblast-like - cells attached to a substrate appear
elongated and bipolar.
(g) Culturing of cells: Cells are cultured as
anchorage-dependent or independent. Cell lines derived from normal tissues are
considered anchorage-dependent and grow only on a suitable substrate, e.g., tissue cells. Suspension cells are anchorage-independent, e.g., blood cells, whereas transformed cell lines either grow as monolayers or as suspension.
(h) Steps:
1. Use sterile technique: The tissue part is
harvested and processed using sterile equipment, reagents, and techniques.
Personal protective equipment is used to avoid contamination. All enzymes and
reagents are filtered sterile condition using a 0.22-micron membrane.
2. Mince/cut tissue: Mince the tissue specimen into
small pieces (usually 2 × 4 mm) with sterile scissors or a scalpel, and then
place the small pieces into the selected buffer, media, or salt solution.
3. Wash and add enzyme: Wash tissue two to three
times to eliminate excess blood proteins and then add enzyme(s) of choice,
likely, collagenase, protease, papain, or trypsin. Usually, about 0.5 to 1.5
mg/ml of selected enzyme is sufficient.
4. Incubation: Further tissue specimen is incubated at
an optimum temperature of 37°C for 30 to 90 minutes with periodic mixing with
the rock specimen.
5. Disperse and wash cells: Cells are dispersed by
gently pipetting them, and then the cell suspension is filtered using a fine mesh.
The cells became settled and decanted excess liquid containing enzymes. Further
cells are washed two to three times with Fetal Bovine Serum (FBS), Bovine Serum
Albumin (BSA), or other inhibitors can also be used to halt enzyme digestion.
6. Resuspension and measure cells: Cells are
resuspended in the correct medium or buffer, and then quantitatively determined
the cell yield and viability. This is an important step in the cell isolation
process to evaluate the result by the dissociation technique. Most researchers have
used a hemocytometer for determining cell yield and trypan blue diazo dye to
measure cell viability.
% of viable cells = Number of unstained cells × 100 /
Total number of cells
(i) Cell toxicity: Cytotoxicity causes inhibition of cell
growth, which is observed an effect on the morphological alteration in the cell
layer or cell shape. It is characterized by abnormal morphology like giant
cells, multinucleated cells, a granular, bumpy appearance, vacuoles in the
cytoplasm or nucleus, etc. Cytotoxicity is determined by substituting materials
such as medium, serum, supplement flasks, etc.
Primary Culture
Cells, when surgically or enzymatically removed from the
organism and placed in a suitable culture environment and grown, are called a primary culture. They have a finite life span and contain a heterogeneous
population of cells. These cells, upon subculture, generate cell lines that have
a limited life span. The lineage of cells that originates from the primary
culture is known as a cell strain. Primary cultures are morphologically similar
to the parent tissues. They have a limited number of cell divisions (Fig. 2)
Fig. 2: Primary cell culture
Established Cell Culture
Primary cell culture, when first subcultured, is known as
secondary cell culture. An established or immortalized cell line is the ability
to proliferate indefinitely by random mutation and artificial modification, such
as artificial expression of the telomerase gene.
Advantages:
• Many kinds of cell lines.
• Generally easy to grow and manipulate.
• Proliferate indefinitely.
• Contact inhibition. Example: HeLa, Sf-9, Cervical cancer.
Transformed Cell Culture
Transformation is the process of conversion of a normal cell
into a cell having some or many of the attributes of a different cell. When a cell
is transformed, the cell loses contact inhibition and becomes immortal. For example,
NIH 3T3 mouse cells are partially transformed and become immortal, but contact inhibition is
inhibited and grow in a monolayer. Transformed cells lack contact inhibition of
movement due to changes in cell surface properties and the loss of many
receptors. They continue to grow and pile up on top of one another as they
proliferate.
Transformed cells are also cultured through a suspension
medium where cells do not attach to the surface of the culture vessels and are
grown in liquid culture medium. Hematopoietic stem cells (derived from blood,
spleen, and bone marrow) and tumor cells grow in suspension much faster, which does not require the frequent replacement of the medium.