THE SUBSTRATE AND CHOICE OF CULTURE VESSEL:-

PRESENTED BY DEBJANI MUKHERJEE ASSISTANT PROFESSOR LPU.

ATTACHMENT & GROWTH: The majority of cells cultured in vitro grow as monolayer on an artificial substrate.
Hence substrate may be correctly charged to allow cell adehesion. Many transformed cell line can be made to grow in suspesion and become independent of the surface charge on the substrate. Most cells need to spread out on a substrate to proliferete.

SUBSTRATE MATERIALS:GLASS:-

Original substrate bec of its optical properties & surface charge. Replaced by plastis (polysterene) which has greater consistency & superior optical properties. Treatment with strong alkali renders glass unsatisfactorily for culture until it is neutralized by an acid wash.

DISPOSABLE PLASTIC:-

Single use sterile polysterene flasks provide simple,reproducible substrate for culture They are of good optical quality & growth surface is flat,providing uniformly distributed & reproducible monolayer culture. Polysterene is the most common cheapest plastic

substrate. Cells can also be grown on PVC, Polycarbonate,thermanox etc.

CHOICE OF CULTURE VESSEL:Several factors govern the choice of culture vessel;

Cell mass required Whether cell grown in suspension or as monolayer. Whether culture should be vented to the atmosphere

or sealed. Frequency of sampling Type of analysis required cost

CELL YIELD: For monolayer culture cell yield is proportional to the

available surface area of the flask. Small volumes & multiple replicates are best perfomed in multiwell plates,which have a no of small wells. Glass bottle are more variable than plastic . Glass bottle should have; One reasonable flat surface. Deep screw cap with good seal & nontoxic liner Shallow sloping shoulders for harvesting monolayer cells after trypsinization & improve effeciency of washing. If large cell yield need special vessel required.

SUSPENSION CULTURE: Cell that grow in suspension can be grown in any types

of flask,plates,or petri dish . Stirrer bottles are used when agitation is required to keep cells in suspension. Agitation is usually by suspended paddle containing magnet,whose rotation is driven by magnetic stirrer. The rotational speed must be kept low to avoid damage from shear stress. Pendulum design is preferable for minimising shear.

VENTING: Multiwell and petri dishes chose for cloning. They have loose fitting lids to give easy acess to dish. They are not sealed & required a humid atmosphere with carbon

dioxide conc controlled. Thin film of liquid may form around the inside of lid ,partially sealing some dishes ,vented lids with molded plastic supports inside used. Perfect seal is done by self adehesive film for multiwell dishes. Flask are vented by slackening the caps when in co2incubator to allow co2 to enter or allow excess co2 to escape in acid producing cell line. Caps with permeable filter are preferred they permit equilibration with gas phase.

UNEVEN GROWTH: Cells can nonuniformly distributed among growth surface. Vibration caused by opening & closing of incubator ,faulty fan motor ,or vibration from equipment can perturb the medium. That result in wave in flask that lead to wave pattern in monolayer creating variation in cell density. Eliminating vibration &minimising entry into incubator help to reduce uneven growth.

COST: Cost has to be balanced against convenience.

Petri dishes are cheaper than flask bt more prone to

infection. Cheap soda glass bottles nt of good optical quality are better than high grade pyrex for culturing bec 2nd one contain lead. Plastic are used than glass.