Culture environment

• Cells do not express the properties characteristic of the same cell type in vivo-cellular environment has been changed

• Cell-cell and cell-matrix interactions are reduced-3D architecture found in in vivo and hormonal and nutritional milieu is altered

• This creates an environment favoring spreading migration and proliferation of of unspecilaised cells

• Influence of env on culture – Nature of substrate or phase on/in cells grow-solid,semisolid,liquid– Physicochemical and physiological constitution of medium– Constitution of gas phase– Incubation temp

Cell adhesion• Cells from solid tissue-grow as adherent monolayers• In subculture before they proliferate the cells have to attach and

spread out• glass having net negative charge; treated plastic• Cell adhesion is mediated by specific cell surface receptors for

molecules in the extracellular matrix• Spreading may be preceded by secretion of extracellular matrix

proteins and proteoglycans by the cells • The matrix adheres to the charged surface –cells bind via specific

receptors• Glass conditioned with previous growth-better surface• Pretreated with matrix constituents such as fibronectin collagen or

gelatin-help to attach and proliferate

Cell adhesion molecules• 3 major classes of transmembrane proteins are involved in cell-cell

and cell-substrate adhesionCell-cell adhesion molecules CAMS (Ca 2+ independent) and cadherins (Ca 2+ dependent)

• Cell substrate interactions are mediated by integrins –receptors for matrix molecules such as fibronectin entactin laminin and collagen

• They bind via specific motif containing arginine-glycine-aspartic acid sequence(RGD)

• Transmembrane proteoglycansCytoskeleton –cell adhesion molecules are attached to

elements of cytoskeleton• Integrins attach to actin microfilaments via linker proteins• Cadherins link to actin cytoskeleton in adherens junctions-

mediating cell shape• Desmosomes also employ cadherins-link to intermediate filaments• Microtubules - cell motility

Cell proliferation

• 4 phases-• M Phase- chromatin condenses into chromosomes and 2 individual

chromatids segregate to each daughter cell• G1 phase- cell either progresses towards DNA synthesis and

another division cycle or exits cell cycle(G0)• S phase- DNA replicates• G2 phase- cell prepares reentry into mitosis

Control of cell proliferation• Low cell density leaves cells with free edges and renders spreading-

permitting entry into cell cycle in presence of mitogenic GF –EGF FGF PDGF

• High cell density inhibits the proliferation of normal cells

Initiation of culture

• A culture is derived by – Outgrowth of migrating cells from a fragment of tissue– Enzymatic or mechanical dispersal of tissue

• Primary culture is followed by uniform cell line• In primary explant technique selection occurs by virtue of cells

capacity to migrate from explant• With dispersed cells by disaggregation technique –only those

survive the disaggregation and adhere to substrate form basis to primary culture

• Primary culture –stage of the culture following isolation of cellsAnd it is a stage before subculture

involves 3 stages– Isolation of tissue– Dissection/disaggregation– Culture following seeding into the culture vessel

Each tissue require diff set of conditions, some common requirements are• Fat and necrotic tissue are best removed during dissection• Tissue should be chopped finely with sharp instruments to cause minimum

damage• Enzymes used for disaggregation should be removed subsequently by gentle

centrifugation• Conc. of cells in primary culture should be much higher than that normally used

for subcultures• A rich medium is preferable to a simple medium-serum: fetal bovine• Embryonic tissue is preferable -disaggregates more readily yields more viable

cells proliferates more rapidly than adult tissue

Isolation of tissue

• Sterilise the site of the dissection with 70% alcohol (eg. Skin)• Remove the tissue aseptically and transfer it to tissue culture lab in

BSS or medium as soon as possible• Do not dissect animals in tissue culture lab –contamination • If there is delay in transfer, can be held at 4C for upto 72h

Chick embryo cell culture• Chick embryos are easier to dissect• Provide mesenchymal cell primary cultures for cell proliferation

analysis• To provide feeder layers• As a substrate for viral propagation• Larger size-easier to dissect out individuals organs to generate

specific cell types-hepatocytes, cardiac muscle and lung epithelium

Protocol

• AIM-Remove embryo aseptically from egg and transfer to dish• Incubate eggs at 38.5C in a humid atmosphere and turn the eggs

through 180C • Swab the egg with 70% alcohol and place it with its blunt end facing

up in a small beaker• Crack the top of shell and peel the shell off to the edge of the air sac

using sterile forceps• Peel off the white shell membrane to reveal the chorioallantoic

membrane CAM below with its blood vessels• Peirce the CAM with sterile curved forceps and lift out the embryo

by grasping it gently under the head• Transfer the embryo to a 9cm petri dish containing 20 ml of DBSS

Primary culture

Primary explant• Harrison and carrel –primary explant technique• Fragment of tissue was embedded in blood plasma or lymph, mixed

with serum and embryo extract –placed on cover slip and inverted over a concavity slide

• Useful for small amounts of tissue such as skin biopsies-cells are lost due to mechanical or enzymatic disaggregation

• Disadvantage- poor adhesiveness of cells• Attaching explants- adherence and migration may be stimulated by

placing a glass coverslip on top of explant with explant near the edge of coverslip

• Plastic dish may be scratched through the explant to attach tissue to the flask

• Treating plastic with polylysine or fibronectin

Primary Explant technique

Tissue in basal salt soln Chopping down to explant size Wash by settling

Remove basal salt solution

Tissue on growth mediaIncubate and change media at intervals

Fresh culture vessel

Enzymatic disaggregation• Cell-cell adhesion in tissues is mediated by glycopeptides which are

calcium dependent(cadherins)-sensitive to EDTA• Integrins-which binf to RGD motif inextracellular matrix- have Ca

binding domains- affected by Ca depletion• Intercellular matrix and basement membranes contain glycoprotein

such as fibronectin and laminin-protease sensitive Enzymes used in enzymatic disaggregation – Trypsin – Collagenase – Elastase – Hyaluronidase – DNase – Pronase (bacterial protease)

• The easiest approach –start with trypsin/trypin-EDTA and adding others proteases to improve disaggregation

Mechanical and enzymatic disaaggregation of tissue • Avoids problems of selection by migration • Yields a higher number of cells representative of tissue• Selection of cells-cells resistant to disaggregation and still capable

of attachment• Embryonic tissue disperses more readily and gives a higher yield of

proliferating cells than adult tissueChoice of trypsin

• purer-less toxic –more predictable its action• Cruder-more effective due to other proteases-most commonly used• Exposure should be minimised-whole tissue at 37C• Dissociated cells should be collected every half an hour

• Removed by centrifugation and neutralised with serum in mediumTwo methods-

• cold trypsinisation –higher yield of viable cells requires less effort• Warm trypsinisation –extensively used

Warm trypsinization• The chopped tissue is washed with dissection basal salt

solutions(DBSS)• Transferred the tissue to a flask containing Trypsin(37 0C).• Contents are stirred at an interval of every 30mins.• Supernatant containing dissociated cells can be collected.• The trypsin is removed by centrifugation after 3-4 hours.• cells are dispersed in a suitable medium and preserved by keeping

the vial on ice.

Warm trypsinization

supernatant

Remove trypsin and centrifuge

Disaggregate cells vials kept on ice

Primary cell culture

• Warm -useful for disaggregation of large amounts of tissue in short time

• Disadvantage-damage due to prolonged exposure to tissue to trypsin at 37C- hence harvesting cells after 30 min in warm trypsin than full time(3-4h)

• To minimise this damage-soak the tissue in trypsin at 4C to allow penetration of enzyme with little tryptic activity

• Cold trypsin method-gives high yeild of viable cells with improved survival after 24h culture

• Preserves more different cell types than warm method• Convenient-as no stirring or centrifugation • Disadvantage –long time and not convenient with large

tissue(greater than 10 g)

Cold trypsinization

Primary cell culture

Other enzymatic procedures• Damaging-eg: some epithelial cells• Ineffective –eg: very fibrous tissue• Collagenase- extracellular matrix with collagen-muscle and

connective tissueEffective for many-embryonic, adult, normal and malignantUseful when too sensitive to trypsin or too fibrous the tissue

• Hyalurodinase and neuraminidase – intracellular adhesion involves carbohydrate

Disaggregation by Collagenase

Collagen is most abundant structural protein in higher animals

Main component in ECM of connective and muscle tissues

Successfully used for the human brain, lung and several epithelial tissues

• Desired tissue suspended in a basal salt solution containing antibiotic• Washed by settling • Incubated in medium containing collagenase for 1-5 days.• Dispersed by pipetting • Cluster of cells are separated by settling• Epithelial and fibroblast cells are separated

Mechanical disaggregation• Primary explant –relatively slow process and highly selective• Enzymatic digestion –more labor intensive, risk of damaging cells• Alternative is mechanical disaggregation

collecting cells that spill when slicedpressing dissected tissue through a series of sievesforcing tissue fragments through a syringe and needlesimply pipetting repeatedly

• Soft tissues such as spleen embryonic liver embryonic and adult brain and some human and animal soft tumors respond well

• Low viability less time• Tissue is not a limitation and efficiency of yield is unimportant

Separation of viable and nonviable cells

• When adherent primary culture is prepared from dissociated cells non viable cells are removed at the first change of medium

• In suspension non viable cells are gradually diluted out when proliferation starts

• By centrifugation on a mixture of Ficoll and sodium metrizoate• Dead cells will form a pellet at bottom