CELL CULTURE
Introduction
The process of removal of cells from
an animal or plant, and their subsequent growth in a favorable artificial environment is referred to as cell culture. The cell to be cultured may
obtained from the tissue directly and disaggregated by enzymatic or mechanical means
before cultivation, or they may be obtained from a cell line or cell strain
that has already been established.
The culture media used to culture animal cells should
deliver nutritional, hormonal, and stromal factors. For the growth and maintenance
of in vitro animal cells, the culture conditions should be similar to in vivo
conditions with respect to temperature, oxygen, and carbon dioxide
concentration, pH, osmolality, and nutrition.
Vertebrate cells can be cultured
using a media-rich in essential amino acids, vitamins, peptides, or protein
growth factors, and frequently provided by serum. Therefore, tissue culture
media used in the previous times were made up of biological fluids such as
plasma, lymph, serum, and tissue extract of embryonic origin. Most of the
cultured vertebrate cells grow when attached to the negatively charged substratum,
identical to the extracellular matrix of animal tissues.
Following types
of cells are involved in the animal cell culture:
1) Primary Cells: These cells are derived from animal
tissues, grow limitedly in culture and give rise to a cell strain.
2) Transformed Cells: These cells are derived from animal
tumors or arise from primary rodent cells, and grow indefinitely in culture.
They have an unstable, aneuploid complement of chromosomes, including abnormal chromosomes. Transformed cells derived
from a single parental cell are known as cell lines.
3) Cultured Cells: These cells are treated with certain
viruses or polyethylene glycol to be fused into heterokaryons (hybrids between
cells of different species, which lose the chromosomes of one species on
dividing).
The culture medium is the most essential factor for
culturing cells and tissues. A cell culture medium is
comprised of the following two parts:
1) Basal Medium: It supports the cellular requirements for nutrients, salts, and pH control.
2) Set of Supplement: It supports other cellular requirements and allows the cells to grow in the basal medium.
Culture
medium provides:
1) Optimum conditions of factors like pH, osmotic pressure,
etc., and
2) Chemical constituents that cannot be synthesized by cells.
Growth of Animal Cells in Culture
Animal cell culturing is more difficult than microbial culturing because animal cells demand more nutrients and should be attached to specially coated surfaces for their growth. Still, there are various undifferentiated and differentiated animal cell types which have been cultured. |
|
The nine amino acids, known as the essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), are not synthesized by adult vertebrate animals and therefore must be obtained from external Apart from these, most of the cultured cells require cysteine, glutamine, and tyrosine also, synthesized by the intact animal and cell, e.g., liver cells synthesize tyrosine from phenylalanine; liver and kidney cells synthesize glutamine. All animal cells both within the organism and in culture can synthesize the remaining eight amino acids; therefore, these amino acids need not be present in the diet or culture medium. Other essential components of media of the animal cell culture are vitamins (cannot make at all or inadequate amounts), various salts, glucose, serum, and the non-cellular part of the blood. Most of the cultured animal cells require special solids surfaces for growth. Within the intact animal tissues, the cells are tightly packed and interact with other cells through the cellular junctions. The cells are also in contact with the extracellular matrix (a complex network made up of secreted proteins and carbohydrates to fill the intercellular spaces). The constituents of matrix secreted by the cells participate in binding the cells in tissues together; as well as it also provides a lattice through which cells can move, especially at the early phase of animal differentiation. In various animal tissues, the extracellular matrices consist of
several common components: l) Fibrous collagen proteins, 2) Hyaluronic acid, 3) A large mucopolysaccharide, 4) Covalently linked polysaccharides 5) Proteins in the form of proteoglycans (mostly carbohydrate), and 6) Glycoproteins.
Given below are the conditions needs to be maintained for the growth of animal cells: l) Balanced Salt Solution (BSS): It is made up of inorganic salts which maintain the osmotic pressure and buffer medium at physiological PH. Some inorganic ions, e.g., Na+, K*, Mg, Ca=2+, CC, S042+, P04, etc., are also required
to stimulate the process Growth. |
3) pH: Neutral pH value (i.e., 7) is considered to be optimum for the growth of cells. It facilitates inspection and control of cultures.
4) Energy Sources: The major source of energy is carbohydrates which initiate growth in cultured cells. The most commonly used sugar is glucose; while others like maltose, sucrose, fructose. Galactose, and mannose are also used.
5) Amino Acids: Arginine, cysteine, histidine, isoleucine, lysine, etc., are the essential amino acids required for the growth of most animal cells.
6) Vitamins: Biotin, choline, folic acid, nicotinic acid, Panthoic acid, pyridoxal, riboflavin, thiamine, and inositol are some of the vitamins required for the growth and multiplication of animal cells.
7) Hormones and Growth Factors: Hormones and growth factors are essential for a variety of different effects on the survival and proliferation of cells. Insulin and hydrocortisone are the most commonly used ones. Also, interleukins, Colony stimulating factors, epidermal growth factors, and fibroblast growth factors are required.
8) Proteins and Peptides: Fetuin, fibronectin, albumin, and transferrin are the protein supplements commonly used for stimulating animal cell proliferation and growth.
9) Fatty Acids and Lipids: These are important components of some serum-free media, but their effect has not been yet established.
10) Accessory Factors: Some additives like iron, zinc, copper, and selenium trace elements also aid in effective cell growth.
General Procedure for Cell Culture
The cell culture methods are either
applied to free-living organisms (bacteria or eukaryotic microorganisms), or to
cells removed from multicellular tissues. Cells can be cultured for a prolonged
time if they split regularly. After that the growth medium is replaced and the
cells are diluted (after first detaching them by trypsin of NaOH from the
support).
For culturing cells efficiently, the Environment in which they found themselves should be maintained before they are
transplanted to the artificial environment (an extracellular fluid derived from
blood). This extracellular fluid is recreated using Ham's tissue culture medium
(commonly for mammalian cells).
Some cells (like those in
bloodstream) survive without attaching to a surface; while others (like those
derived from solid tissues) require a special surface. There are also cells
like yeast and some bacteria types which survive under either condition and exhibit
different phenotypes depending on whether or not they are attached to a
surface. In most of the cells derived from tissues, nutrients are provided by a
liquid broth that washes the cells attached to a surface. Some cells require an
"air-liquid interface" for their growth; the cells, in this case, are
grown on a raft of organic material floating on the surface of a nutrient broth
and acting like a wet sponge to feed the cells from underneath while the tops
are exposed to the air.
For bacteria
and yeast, small quantities of cells are grown on a Solid support enriched with
nutrients embedded in its stiff nutritious Jello, while large-scale cultures
are grown with the cells suspended in a nutrient broth.
A population
of cells derived from a single parental cell is called a clone, which
may be derived from continuous cell lines or from primary culture. Cloning
within a cell population reduces the degree of genetic and phenotypic
variation.
Primary Cell Culture
The surgically removed cells of an
organism attach, divide. and grow when placed into a suitable culture
environment: the resultant culture is the primary culture. These cultures are
obtained from intact or dissociated tissues or organ fragments. After a primary
culture is sub-cultured, it is termed as cell line. This can be done by the
following two ways:
l) Explant Cultures: In this
method, small pieces of tissue attached to a glass or to a treated plastic
culture vessel are bathed in a culture medium. After a few days. the cells move
out from the tissue explant onto the culture vessel surface or substrate for
their division and growth.
2) Enzymatic Dissociation:
This method is the more widely used one and also quick as it involves adding
digesting enzymes (e.g., trypsin or collagenase) to the tissue fragments so
that the cement holding the cells together dissolves. Thus, a suspension of
single cells is formed which is then placed in culture vessels and allowed to
divide and grow.
Culturing of Animal Cells
Primary cultures do not contain
variety of cells present in the tissue because they cannot attach to the
substrate and survive. The dead cells get easily eliminated when the medium is
poured out during sub-culture. After some time, the available substrate is
occupied by the monolayer. Then the cells are harvested from primary culture.
l) Capacity for Biotransformation:
In many cases, primary cell culture metabolism has greater similarity to in
vivo than that to sub-cellular fractions used as an exogenous source for
biotransformation.
2) Tissue-Specific
Functions: The primary culture retains tissue-specific functions. For
example, on exposing primary cultures of rat myocardial cells (having
synchronously beating cells) to tricyclic cardiotoxic antidepressants, beating
is observed.
The main disadvantages
of the primary cultures are the necessity to isolate cells for each experiment,
and the procedure followed for isolation disrupts the tissues, along with
proteolytic enzymes. Thus, specific membrane receptors are damaged or lost, integrity
of the membrane is damaged, and cellular products
are lost.
for more pharmacy notes:
No comments:
Post a Comment