lecture5 - nutritional and substrate reqs of cell culture

8/14/2019 Lecture5 - Nutritional and Substrate Reqs of Cell Culture

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Nutritional and substrate reqs

of cell culture

Maulik P. Suthar


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Minimal Requirement for Growing Cells

Artificially

The nutrient media used for culture of animal

cells and tissues must be able to support their

survival as well as growth, i.e., must provide

nutritional, hormonal and stromal factors. The various types of media used for tissue

culture may be grouped into two broad

categories:

1. Natural Media and

2. Artificial Media


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Natural Media

Clots. The most commonly used clots are plasma clots which have been inuse for a long time. Plasma is now commercially available either in liquidor lyophilized state. usually from the blood of male fowl, but blood clottingmust be avoided during the preparation.

Biological Fluids. Of the various biological fluids used as culture medium

(e.g., amniotic fluid, ascitic and pleural fluid, aqueous humor from eye,insect haemolymph, serum etc.)

serum is the most widely used. Serum may be obtained from adult humanblood, placental cord blood, horse blood or calf blood (foetal calfserum, newborn calf serum, and calf serum);of these foetal calf serum isthe most commonly used. Serum is the liquid exuded from coagulating

blood. Different preparations of serum differ in their properties; they have tobe tested for sterility and toxicity before use.

Tissue Extracts.Chick embryo extract is the most commonly used tissueextract, but bovine embryo extract is also used. Other tissue extracts thathave been used are spleen, liver, bone marrow, leucocytes etc. extracts.Tissue extracts can often be substituted by a mixture of amino acids andcertain other organic compounds.


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Artificial Media

purposes:

(2) immediate survival (a balanced salt solution,

with specified pH and osmotic pressure is

adequate),(3) prolonged survival (a balanced salt solution

supplemented with serum, or with suitable

formulation of organic compounds),

(4) indefinite growth

(5) specialized functions


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Serum Containing Media

The various defined media, e.g., Eagles

minimum essential medium etc.

When supplemented with 5-20% serum

are good nutrient media for culture of most

types of cells.

The serum provides various plasma

proteins, peptides, lipids, carbohydrates,

minerals, and some enzymes.


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Serum Containing Media -functions

1. It provides the basic nutrients for cells; the nutrients are present both in thesolution as well as are bound to the proteins.

2. It provides several hormones, e.g., insulin, which is essential for growth ofnearly all cells in culture, cortisone, testosterone, prostaglandin etc.

3. It contains several growth factors, e.g., platelet derived growth factor(PDGF), transforming growth factor P (TGF-p), expidermal growth factor,

fibroblast growth factor, endothelial growth factor etc.; these are present inconcentrations of\igfl. Both hormones and growth factors are involved in growth promotion and

specialized cell function. A given hormone or growth factor may stimulategrowth of one cell type, may have no effect on another and may even beinhibitory to some others.

PDGF induces proliferation in fibroblasts but induces differentiation of some

types of epithelia. proliferation of a single cell type may be induced by more than one growth

factor, e.g., fibroblasts respond to PDGF, epidermal growth factor, fibroblastgrowth factor and somatomidins.


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Serum Containing Media

4. A major role of serum is to supply proteins, e.g., fibrobnectin, whichpromote attachment of cells to the substrate. It also provides spreadingfactors that help the cells to spread out before they can begin to divide.Although cells do produce these factors, but typsinized cells are usuallyunable to attach to the substrate.

5. It provides several binding proteins, e.g., albumin, transferrin, which carry

other molecules into the cell. For example, albumin carries into cells lipids,vitamins, hormones etc. Transferrin usually carries Fe in a nonbasic form,but binding of transferrin to its receptor in cell membrane is believed to bemitogenic.

6. It increases the viscosity of medium and, thereby, protects cells frommechanical damages, e.g., shear forces during agitation of suspensioncultures.

7. Protease inhibitors present in the serum protect cells, especially trypsinisedcells, from proteolysis.

8. The serum also provides several minerals, e.g., Na+, K+,Fe2+, Zn2+, Cu2+,etc.

9. It also acts as a buffer.


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GROWTH FACTORS

Growth factors are proteins that bind to

receptors on the cell surface, with the

primary result of activating cellular

proliferation and/or differentiation.

Many growth factors are quite versatile,

stimulating cellular division in numerous

different cell types; while others arespecific to a particular cell-type.


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GROWTH FACTORS

Cytokines are a unique family of growth factors. Secreted primarilyfrom leukocytes, cytokines stimulate both the humoral and cellularimmune responses, as well as the activation of phagocytic cells.

Cytokines that are secreted from lymphocytes are termedlymphokines, whereas those secreted by monocytes ormacrophages are termed monokines.

A large family of cytokines are produced by various cells of thebody.

Many of the lymphokines are also known as interleukins (ILs),since they are not only secreted by leukocytes but also able to affectthe cellular responses of leukocytes.

Specifically, interleukins are growth factors targeted to cells ofhematopoietic origin.


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GROWTH FACTORS


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Buffer compositions


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PBSA

PBSA is used as a washing solution

before disaggregation, and as a diluent for

trypsin (trypsin is used at 2.5 g, crude

preparation, or 0.1 g, purified by repeatedcrystallization, per litre). PBSA is also

used as the base solution for preparing

EDTA solution (Na2 EDTA.2H2O at 372mg/l, 1 mM) which is autociaved before

using.


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Disadvantages of using serum

1. Serum may inhibit growth of some cell types, e.g., epidermalkeratinocytes.

2. Serum may contain some cytotoxic or potentiallycytotoxicconstituents. For example, foetal calf serum contains theenzyme polyamine oxidase which converts polyamines likespermidine and sperrnine (secreted by fast growing cells) into

cytotoxic polyaminoaldehydes.3. There is a large variation in serum quality from one batch to

another; this requires costly and time consuming testing every timea new batch has to be used.

4. Some growth factors may be inadequate for specific cell typesand may need supplementation.

5. It interferes with downstream processing when cell cultures areused for production of biochemicals.

6. The supply of serum is always lower than its demand.


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Serum-Free Media -advantages

1. Improved reproducibility of results from differentlaboratories and over time since variation due to batchchange of serum is avoided.

2. Easier downstream processing of products from

cultured cells.3. Toxic effects of serum are avoided.

4. Biassays are free from interference due to serumproteins.

5. There is no danger of degradation of sensitive proteinby serum proteases.

6. They permit selective culture of differentiated andproducing cell typesfrom the heterogenous cultures.


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Serum-Free Media - disadvantages

1. Most serum-free media are specific to one cell type.Therefore, different media may be required for differentcell lines.

2. Reliable serum-free preparations, for most of the mediaformulations are not available commercially. Thisnecessitates time consuming task of preparing thedesired formulations in the laboratory.

3. A greater control of pH, temperature etc. is necessaryas compared to that with serum containing media.

4. Growth rate and the maximum cell density attainedare lower than those with serum containing media.

5. Cells tend to become fragile during prolonged agitatedcultures unless biopolymers or synthetic polymers areadded.


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Chemically Defined Media: These media contain

contamination- free ultra-pure inorganic and

organic constituents, and may contain pure

protein additives, like insulin, epidermal growthfactor etc. that have been produced in bacteria

or yeast by genetic engineering.

Protein-free Media. In contrast, protein-free

media do not contain any protein; they onlycontain non-protein constituents necessary for

culture of the cells.


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BALANCED SALT SOLUTIONS

Balanced salt solutions contain an array of inorganicionic species that are essential for critical functions whencells are removed from theirin vivo milieu. These include

(1) sodium (Na*) and potassium (K+) to regulate tonicity

and permeability,(2) calcium (Ca2*) and magnesium (Mg2+) to maintain the

integrity of cell membranes and internal structures

(3) phosphate (HPOj-HjPOJ) and/or bicarbonate (HCO^) tocontrol the hydrogen ion concentration through theirbuffering effect. Most balanced salt solutions also includeglucose, which provides a readily available energysource for cells.


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Preparation of Balanced Salt Solutions

Balanced salt solutions that are filter-sterilized

and ready for use can be purchased from

various commercial suppliers.

Sterile liquid concentrates (10X) can also beobtained that are diluted with nine parts of

sterile, distilled water before use.

These concentrated salt solutions do not contain

NaIIC03 and it must be added from a sterile

stock solution.


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Preparation of Balanced Salt Solutions

Powdered salt mixtures. Balanced salt solutions can be preparedfrom dry mixtures of chemicals that have been powdered in a ballmill. They are available commercially.

These mixtures do not contain NaHCO3; anhydrous MgSO4 issubtituted for the magnesium salt in the formulation.

The powdered mixture is dissolved in distilled water, NaHC03 isadded, and the salt solution is sterilized by pressure filtrationthrough a 0.22-jum membrane (Millipore Corporation,Waltham,ass.).

Vacuum filtration should not be used, because C02 will be drawn offand salt solution will become abnormally alkaline.

The salt solution is dispensed into sterile screwcapped prescriptionbottles of a convenient size and storedat 4c.


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Preparation and Sterilization of Medium

The various media constituents and other reagents used in cellcultures must be carefully sterilized either by autoclaving or byfiltration. Heat stable constituents tike water, salts, supplements likepeptone or tryptose etc. are autoclaved at 121C for 20 min.

But heat labile constituents like serum, trypsin, proteins, growthfactors etc. must be sterilized by filtration through a 0.2 mm porosity

membrane filter. Each filtrate should be tested for sterility to avoidfailure due to contamination.

In case of soda glass, caps should be left slack to avoid breakingduring autoclaving.

Autoclaving is preferred to filtration since it is cheaper, needs lesslabour and is uniformly effective. Therefore wherever possible,

autoclaving should be resorted to and autoclavable versions of themedia should be used. Most of the media, however, now availablecommercially are usually presterilized.

In artificial culturing of cells, Isolation of explants plays an importantrole.


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Isolation of Explants

Explants should be dissected out following an appropriate protocol developed to avoid contamination and retain cell survival. In general, embryos and other contents of uterus are free from contamination, while in adult tissues bacterial contamination may be common. In simple terms, the site of

opening is sterilized with 70% alcohol, the tissues are removed aspectically, and usually placed in a balanced salt solution, e.g., BBSS or HBSS, supplemented with antibiotics (Table 3.1 footnote 2). All subsequent handling should be under strictly aseptic conditions, preferably under laminar-flow cabinets or

hoods or in sterile rooms. Care should be taken not to violate ethical or legal boundaries in obtaining the desired explant.


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Disaggregation of Explants

Cell cultures are generally started from disaggregated explants. Occasionally, cells do migrate out of the cultured whole tissues; such

cells are then separated by trypsin or other enzymatic treatment toestablish cell cultures. But in general, the tissues are first disaggregatedin one of the following ways:

(3) Mechanical disaggregation,

(4) enzymatic disaggregation, and(5) EDTA treatment.

Mechanical Disaggregation. The tissue is chopped into fairly small pieces, say,of 1 mm3 in size. These are then cultured into a suitable vessel; thepieces attach to the substrate either due to their own adhesiveness, thedish may be scratched to facilitate attachment, or clotted plasma may beused. Cells grow out from the tissue pieces which are trypsinized andsubcultured .

The explants themselves may be retained in the same dish or transferred to anew vessel to obtain further cell growth.


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Enzymatic Disaggregation.

(0.25% crude or 0.01-0.05% pure) and collagenase (200-2000units/ml, crude) are the most commonly used enzymes, but otherenzymes like elastase, mucase, pappain etc. have also been used.

Trypsin is preferred because it is effective for many tissues, istolerated by most cells, and its residual activity is neutralized byserum (or by a tryspin inhibitor in case of serum-free media). The

dissociated cells are collected, washed free of the enzyme andcultured.

In some cases, trypsin may be either damaging, e.g., epithelial cells,or may be ineffective, e.g., fibrous tissues.

Therefore, disaggregation of several normal and malignant tissuesis better achieved by collagenase which readily digests away

matrices containing collagen. The trypsin and collagenase solutions are generally prepared in a

balanced salt solution .


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EDTA Treatment

Some tissues like epithelium require Ca2+ and Mg2+ions for their integrity.

Disaggregation of such tissues is readily achieved by atreatment with EDTA (ethylene-diaminetetra-acetic acid)

prepared in a balanced salt solution This treatment isgenerally used for separation of cells from establishedcultures of epithelial tissues.

Enzymatic disaggregation generally gives higher cellyields than the mechanical method, but is more selective

since only certain cell types will survive dissociation.Therefore, in practice, often the tissues are only reducedto small clusters of cells by an enzyme like collagenase,and these clusters are used to initiate cell cultures.

lecture5 - nutritional and substrate reqs of cell culture

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