Cell Isolation Theory

Tissue Types

This section summarizes the general characteristics of extracellular matrices associated with various types of tissue. Coupled with the descriptions of individual enzymes offered in the next section, this information will aid in choosing the enzyme(s) best suited for a particular tissue.

Epithelial Tissue

In the adult, epithelium forms such tissues as the epidermis, the glandular appendages of skin, the outer layer of the cornea, the lining of the alimentary and reproductive tracts, peritoneal and serous cavities, and blood and lymph vessels (where it is usually referred to as "endothelium"). Structures derived from outpouchings from the primitive gut, including portions of the liver, pancreas, pituitary, gastric and intestinal glands, are also composed of epithelial tissue.

Epithelial cells are typically packed so closely together that there is very little intercellular material between them. An extremely tight bond exists between adjacent cells making dissociation of epithelium a difficult process.

On the lateral surfaces of adjacent epithelial cells there are four distinct types of intercellular bonds: the zonula occludens, zonula adherens, macula adherens and nexus . The former three are often closely associated to form a junctional complex. In the zonula occludens , or "tight junction", there are multiple sites of actual fusion of the adjacent unit membranes interspersed by short regions of unit membrane separation of approximately 100-150 Å. In a zonula adherens , or "intermediate junction", a fine network of cytoplasmic filaments radiates from the cell membrane into the cytoplasm. The space between unit membranes of adjacent cells is approximately 150-200 Å and is composed of an intercellular amorphous substance of unknown composition. In the macula adherens , or "desmosome", there is a somewhat similar array of intracellular filaments. The adjacent unit membrane space is approximately 150-200 Å and consists of an extracellular protein and glycoprotein ground substance, often with an electron-dense bar visible within it. The integrity of the desmosome requires calcium, and it is broken down by EDTA and calcium-free media. The enzymes collagenase, trypsin and hyaluronidase can also dissociate the desmosome. The nexus , or "gap junction", covers most of the epithelial cell surface. In these areas, the unit membranes appear tightly attached and are separated by only 20 Å. The intercellular material consists of an amorphous, darkly-staining substance.

On the basal surface of the epithelium where it overlays connective tissue, there is an extracellular bonding layer or sheet called the basal lamina. The lamina is composed of a network of fine, collagen-like reticular fibers embedded in an amorphous matrix of high and low molecular weight glycoproteins.

Connective Tissue

Connective tissue develops from mesenchymal cells and forms the dermis of skin, the capsules and stroma of several organs, the sheaths of neural and muscular cells and bundles, mucous and serous membranes, cartilage, bone, tendons, ligaments and adipose tissue.

Connective tissue is composed of cells and extracellular fibers embedded in an amorphous ground substance and is classified as loose or dense, depending upon the relative abundance of the fibers. The cells, which may be either fixed or wandering, include fibroblasts, adipocytes, histiocytes, lymphocytes, monocytes, eosinophils. neutrophils, macrophages, mast cells, and mesenchymal cells.

There are three types of fibers: collagenous , reticular , and elastic , although there is evidence that the former two may simply be different morphological forms of the same basic protein. The proportion of cells, fibers and ground substance varies greatly in different tissues and changes markedly during the course of development.

Collagen fibers are present in varying concentrations in virtually all connective tissues. Measuring 1-10 µm in thickness, they are unbranched and often wavy, and contain repeating transverse bands at regular intervals. Biochemically, native collagen is a major fibrous component of animal extracellular connective tissue; skin, tendon, blood vessels, bone, etc. In brief, collagen consists of fibrils composed of laterally aggregated polarized tropocollagen molecules (M.W. 300,000). Each rod-like tropocollagen unit consists of three helical polypeptide a-chains wound around a single axis. The strands have repetitive glycine residues at every third position and an abundance of proline and hydroxyproline. The amino acid sequence is characteristic of the tissue of origin. Tropocollagen units combine uniformly in a lateral arrangement reflecting charged and uncharged amino acids along the molecule, thus creating an axially repeating periodicity. Fibroblasts and possibly other mesenchymal cells synthesize the tropocollagen subunits and release them into the extracellular matrix where they undergo enzymatic processing and aggregation into native collagen fibers. Interchain cross-linking of hydroxyprolyl residues stabilizes the collagen complex and makes it more insoluble and resistant to hydrolytic attack by most proteases. The abundance of collagen fibers and the degree of cross-linking tend to increase with advancing age, making cell isolation more difficult.

Reticular fibers form a delicate branching network in loose connective tissue. They exhibit a regular, repeating subunit structure similar to collagen and may be a morphological variant of the typical collagen fibers described above. Reticular fibers tend to be more prevalent in tissues of younger animals.

Elastic fibers are less abundant than the collagen varieties. They are similar to reticular fibers in that they form branching networks in connective tissues. Individual fibers are usually less than 1 µm thick and exhibit no transverse periodicity. The fibers contain longitudinally-arranged bundles of microfibrils embedded in an amorphous substance called elastin. Like collagen, elastin contains high concentrations of glycine and proline, but in contrast has a high content of valine and two unusual amino acids, desmosine and isodesmosine. Fibroblasts and possibly other mesenchymal cells synthesize the elastin precursor, tropoelastin, and release it into the extracellular matrix where enzymes convert the lysine residues into the desmosines. Polymerization of elastin occurs during interchain cross-linking of the latter. In this state, elastin is very stable and also highly resistant to hydrolytic attack by most proteases.

The viscous extracellular ground substance in which connective tissue cells and fibers are embedded is a complex mixture of various glycoproteins, the most common being hyaluronic acid, chondroitin sulfate A, B, and C and keratin sulfate. Each of these glycoproteins is an unbranching polymer of two different alternating monosaccharides attached to a protein moiety. Hyaluronic acid, for example, contains acetyl glucosamine and glucuronate monomers and about 2% protein, while the chondroitin sulfates contain acetyl galactosamine and glucuronate or iduronate monomers and more than 15% protein. The relative abundance of these glycoproteins varies with the origin of the connective tissue.

Dissociating Enzymes

While many enzyme systems have been investigated by researchers performing cell isolations, the enzymes discussed here have been found satisfactory for a wide variety of tissues from many different species of various ages.

Collagenase

Bacterial collagenase is a crude complex containing a collagenase more accurately referred to as clostridiopeptidase A which is a protease with a specificity for the X-Gly bond in the sequence Pro-X-Gly-Pro, where X is most frequently a neutral amino acid. Such sequences are often found in collagen, but only rarely in other proteins. While many proteases can hydrolyze single-stranded, denatured collagen polypeptides, clostridiopeptidase A is unique among proteases in its ability to attack and degrade the triple-helical native collagen fibrils commonly found in connective tissue.

True collagenase may cleave simultaneously across all three chains or attack at a single strand. Mammalian collagenases split collagen in its native triple-helical conformation at a specific site yielding fragments, TC A and TC B, representing 3/4 and 1/4 lengths of the tropocollagen molecule. After fragmentation the pieces tend to uncoil into random polypeptides and are more susceptable to attack by other proteases.

Bacterial collagenases are usually extracted from host invasive strains. These enzymes differ from mammalian collagenases in that they attack many sites along the helix. Collagenases from Clostridium histolyticum , first prepared by Mandl, et al., have been most thoroughly studied. Commercially available collagenase has been limited primarily to that from Cl. histolyticum ; although, other sources have recently become available. Clostridial collagenase also degrades the helical regions in native collagen preferentially at the X-Gly bond in the sequence Pro-X-Gly-Pro where X is most frequently a neutral amino acid. This bond in synthetic peptide substrates may also be split.

Purified clostridiopeptidase A alone is usually inefficient in dissociating tissues due to incomplete hydrolysis of all collagenous polypeptides and its limited activity against the high concentrations of non-collagen proteins and other macromolecules found in the extracellular matrix. The collagenase most commonly used for tissue dissociation is a crude preparation containing clostridiopeptidase A in addition to a number of other proteases, polysaccharidases and lipases. Crude collagenase is well suited for tissue dissociation since it contains the enzyme required to attack native collagen and reticular fibers in addition to the enzymes which hydrolyze the other proteins, polysaccharides and lipids in the extracelluar matrix of connective and epithelial tissues.

The first commercially available collagenase was offered by Worthington in 1959. At that time we offered one type of crude enzyme which we tested only for collagenase activity. Eventually, with the cooperation of many in the research community, four basic profiles were identified:

Type 1 containing average amounts of assayed activities (collagenase, caseinase, clostripain, and tryptic activities). It is generally recommended for epithelial, liver, lung, fat, and adrenal tissue cell preparations.

Type 2 containing greater clostripain activity. It is generally used for heart, bone, muscle, thyroid and cartilage.

Type 3 selected because of low proteolytic activity. It is usually used for mammary cells.

Type 4 selected because of low tryptic activity.. It is commonly used for islets and other applications where receptor integrity is crucial.

Correlations between type and effectiveness with different tissues have been good, but not perfect, due in part to variable parameters of use. Nevertheless most researchers consider the tissue-typing of crude collagenase lots to be a valuable service. A detailed description of the Worthington collagenase assay as well as our procedure for Clostridiopeptidase A testing can be found in the Worthington Enzyme Manual .

Trypsin

Trypsin is a pancreatic serine protease with a specificity for peptide bonds involving the carboxyl group of the basic amino acids, arginine and lysine. Trypsin is one of the most highly specific proteases known, although it also exhibits some esterase and amidase activity.

Purified trypsin alone is usually ineffective for tissue dissociation since it shows little selectivity for extracellular proteins. Combinations of purified trypsin and other enzymes such as elastase and/or collagenase have proven effective for dissociation.

"Trypsin" is also the name commercial suppliers have given to pancreatin, a crude mixture of proteases, polysaccharidases, nucleases and lipases extracted from porcine pancreas. NF 1:250, a commonly used "trypsin" preparation, has the potency to bring about the proteolytic digestion of 250 times its weight of casein under assay conditions specified by the National Formulary. It is important to realize that this assay procedure is not specific for trypsin, although pancreatin does contain this enzyme. Nomenclature notwithstanding, crude "trypsins" like NF 1:250 and 1:300 are widely used for dissociating tissues, perhaps because the tryptic and contaminating proteolytic and polysaccharidase activities do bring about a preferential attack of the extracellular matrix. It appears, however, that crude trypsin and crude collagenase dissociate tissues by different mechanisms, and difficulties are often encountered when using NF 1:250 preparations -- the most common being incomplete solubility, lot-to-lot variability, cell toxicity, and cell surface protein/receptor damage.

In tissue culture laboratories, researchers use purified trypsin to release cells into suspension from monolayers growing on the interior surfaces of culture vessels. Most cells originating from normal tissues and not highly adapted to artificial culture conditions grow in monolayers, i.e., a layer of cells one cell thick adhering to the interior surface of the culture vessel. Because such cells are more like cells in normal tissues, many tissue culture researchers are studying cells that grow in monolayer culture.

Monolayer cultures are commonly grown in glass or polystyrene roller bottles, culture flasks, or Petri dishes. Plastic vessels used in tissue culture work are specially treated to ensure good adherence of cells to the vessel walls. For a detailed discussion of cell harvesting, see page xv of this guide.

Some of the most frequently used grades of purified trypsin for cell isolation procedures are the Worthington product Codes: TL, TRL, TRLS, and TRLVMF. These products are suitable for cell harvesting as well as tissue dissociation.

Elastase

Pancreatic elastase is a serine protease with a specificity for peptide bonds adjacent to neutral amino acids. It also exhibits esterase and amidase activity. While elastase will hydrolyze a wide variety of protein substrates, it is unique among proteases in its ability to hydrolyze native elastin, a substrate not attacked by trypsin, chymotrypsin or pepsin. It is produced in the pancreas as an inactive zymogen, proelastase, and activated in the duodenum by trypsin. Elastase is also found in blood components and bacteria.

Because elastin is found in highest concentrations in the elastic fibers of connective tissues, elastase is frequently used to dissociate tissues which contain extensive intercellular fiber networks. For this purpose, it is usually used with other enzymes such as collagenase, trypsin, and chymotrypsin. Elastase is the enzyme of choice for the isolation of Type II cells from the lung.

Hyaluronidase

Hyaluronidase is a polysaccharidase with a specificity for endo-N-acetylhexosaminic bonds between 2-acetoamido-2-deoxy-beta-D-glucose and D-glucuronate. These bonds are common in hyaluronic acid and chondroitin sulfate A and C. Because these substances are found in high concentrations in the ground substance of virtually all connective tissues, hyaluronidase is often used for the dissociation of tissues, usually in combination with a crude protease such as collagenase.

Papain

Papain is a sulfhydryl protease from Carica papaya latex. Papain has wide specificity and it will degrade most protein substrates more extensively than the pancreatic proteases. It also exhibits esterase activity.

With some tissues papain has proved less damaging and more effective than other proteases. Huettner and Baughman (1986) describe a method using papain to obtain high yields of viable, morphologically intact cortical neurons from postnatal rats.

Chymotrypsin

Chymotrypsin is a protease which preferentially catalyzes the hydrolysis of peptide bonds involving the aromatic amino acids tyrosine, phenylalanine, and tryptophan. In addition it acts upon the peptide bonds of leucyl, methionyl, asparagenyl and glutamyl residues, and the amides and esters of susceptible amino acids.

Chymotrypsin is used to a limited extent in tissue dissociation, usually in combination with trypsin and elastase.

Deoxyribonuclease I

Often as a result of cell damage, deoxyribonucleic acid leaks into the dissociation medium increasing viscosity and causing handling problems. Purified deoxyribonuclease is sometimes included in cell isolation procedures to digest the nucleic acids without damaging the intact cells.

Neutral Protease (Dispase)

Neutral Protease (Dispase) is a bacterial enzyme produced by Bacillus polymyxa that hydrolyses N-terminal peptide bonds of non-polar amino acid residues and is classified as an amino-endopeptidase. Its mild proteolytic action makes the enzyme especially useful for the isolation of primary and secondary (subcultivation) cells since it maintains cell membrane integrity.

Neutral Protease (Dispase) is also frequently used as a secondary enzyme in conjunction with collagenase and/or other proteases in many primary cell isolation and tissue dissociation applications. Neutral Protease (Dispase) dissociates fibroblast-like cells more efficiently than epithelial-like cells so it has also been used for differential isolation and culture applications. Other advantages are its non-mammalian (bacterial) source and its ability to be inhibited by EDTA.

Trypsin Inhibitor (soybean)

The trypsin inhibitor from soybean inactivates trypsin on an equimolar basis; however it exhibits no effects on the esterolytic, proteolytic or elastolytic activities of porcine elastase. Cell isolation procedures occasionally call for a trypsin inhibitor, usually the inhibitor from soybean (Worthington code SIC ).