Lecturer (IAEA expert mission)

Lecture Topic: Keratinocyte Culture

Limitation of the skin transplantation - as an autograft

	Limited donor site
	Inability to harvest from critically injured patients
	Contamination problem is highly hazardous for the keratinocyte cell growth
	Limited availability in a given period of time ( not adequate area of one sheet for covering sites of patient)
	Patients physiological acceptance (under stress condition)

Application Potential

Even 60% burn patients also can cure using KCC sheets. Usually for the treatments of burn patients, surgeons use to transplant patients own skin layers from different body locations. But it is limited for long period of treatments. Therefore we would be better to search for alternative ways. KCC is more sophisticated set of laboratory techniques can adopt with purified and specific chemical reagents. Growing cells and manage them upto safe clinical application is comes under ultra modern advances of cell and molecular biology and cell culture techniques including surgery and immunology.

Specificity and sensitivity of skin culture technology (KCC)

Overall process completely depend on the level of hygiene or contamination. Therefore environmental management for such a patient is menace. Application of KCC sheets needed more technical and scientific guidance. It has a direct impact on wound healing. Growing cells are highly sensitive for toxic substances. Medium preparation has to be done very carefully with the knowledge of each and every substances chemical composition, purity, nature and stability. Sterility and the specificity are the most important properties of this entire process.

Current status and further development of cell culture

This modern practice is very commonly using in Europe and USA. In the field of transplantation of human cells and tissues from one human to another is now more developed. Currently tissue culture technique diversified upto stem cells, cartilage cells, sperm cells etc.

Keratinocyte Cell Culture

Keratinocyte autografts were first used to treat burn victims in 1981. Small biopsies from burned patients were cultured in vitro and these cultured autografts were subsequently placed on small full-thickness wounds on the arms of the same patients. Successful re-epithelialization of the wound occurred in six weeks. Cultured autografts were subsequently used to provide partial coverage of very extensive burns (over 95% of the body surface area and have been life saving in some instances (Hansbourough JF 1990).

Three methods of initiating a culture

Organ Culture

Tissue are cultured at the liquid gas interface, which favors retention of spherical or three-dimensional shape. This implies that the architecture characteristics of the tissue in vivo is retain.

Primary explant culture

Fragment of tissue is placed at a glass or plastic liquid interface where, following attachments, migration is promoted in the plane of the solid substrate. Cell culture implies that the tissue or out growth from the primary explant, is dispersed (mechanically or enzymatically) into a cell suspension, which they then be cultured as an adherent monolayer on a solid substrate or as a suspension in the culture medium.

Cell culture

The formation of cell culture from a primary culture implies increase in local cell number over several generations and that cells or lineage's with similar high growth capacity will predominant resulting in a degree of uniformity in the cell population. The line may be characterized, and those characteristics will apply for most of its finite life span.

Advantages and disadvantages of biologic skin substitutes

Laboratory chemicals and reagents

This will results better and more confidant results if we purchase the same laboratory made chemicals for the activity. Culture stability and the reliability get through more precision with the outcome. Therefore attention and the safety precautions pointed out.

Exclusion of Antigen Presenting Cells (APC) and Langahan Cells (immunogenicity).

All three types of epidermal cells (langerhans cells, melanocytes, Merkel cells) repopulate cultured epithelial autografts during the first postgrafting year (Tania 1993). “We have recently obtained additional biopsies of cultured epithelial autografts at 3-8 years post grafting, bringing the total number of long term followup biopsies to 39 from a total of 10 pediatric patients (Tania 1993)”.

According to the dermal regeneration on long term follow-up, remodeling of the collagen to the bilayered distribution and vascular remodeling to form a superficial dermal plexus with capilary arcades show considerably better results with respect to the features of partial regeneration with the addition of elastin expression (Tania 1993).

Application of radiation is important with much attention to the molecular and cellular aspects of feeder cell line preparation and Growth arrest (G phase). Therefore it can easily and safely establish feeder cell line preparation with radiation other than chemical methods. Radiation or chemical treatment will inhibit the cell proliferation by arresting the cell cycle.

Standard Operating Procedure (SOP) for practical work

	Turn on the LAF cabinet (keep minimum 45 minutes before use). 70% alcohol can be used as a surface sterilant.
	Select proper instrument and arrange the table. Instruments have to be small and unique. Eye surgery instruments are better.
	Carefully take out the sample from the container.
	Check the sample visually and wash it properly with antiseptic treated solutions. It is easy to use five containers for this step with adequate amount of washing solutions. This step can be done even after separation the skin sample into small pieces.
	Spread the skin on a petridish. Dissect the additional fat tissues from the skin. Divide the sample into three centrifuge tubes (15ml).
	Add 2N 0.5% tripsin into each and every tube. And add the same volume of diluting buffer (PBS) for every tube.
	Incubate 370C for 0.5 hrs (5-10% CO2 preferred) (warm tripsinization).
	Take out the cloudy sample.
	Add more tripsin and keep another 30 minutes inside the incubator.
	Filter the solution using gauze sieve.
	Filter the solution using gauze sieve. Transfer to centrifuge tubes and centrifuge 12 X 100 rpm and take out the sample and add FBCS and DMEM.
	Cell counting (cell culture is depend on initial cell density. Optimal density require for the establishment of the culture. Take the cell suspension into aliquots. Mixed with triptopan blue (1:1). Put a drop of solution on the haemocytometer (1/10 thickness) and the coverslip. Observe and take the cell count through the inverted microscope (and counter).

Cell number is depending on the effectiveness of cell line preparation. It is very rarely depend on the cell origin and the cell type. When the cell number is higher, result will make difficult to the cell multiplication and layers formation.

Calcium free media and buffers

Three major classes of transmembrane proteins have been shown to be involved in cell-cell and cell-substrate adhesion. Cell-cell adhesion molecules, CAMs (Ca²⁺ independent), and cadherins (Ca²⁺ dependant) are involved primarily interactions between homologous cells. They are self-interactive, like molecules in opposing cells interact with each other (Edelman, 1986, 1988: Rosenman and Gallatin, 1991). Cell substrate interactions are mediated primarily by intergrins, receptors for matrix molecules such as fibronectin, lamin and collagen, which bind to them via specific motif usually containing the RGD (arginine, glycine, aspartic acid) sequence Yamada, 1991).

Documentation

Standard operating procedures and work instructions needed for routinely working KCC laboratories. Each and every step of KCC procedures will practice in a standardize manner. Laboratory standardization and procedures validation needed for the KCC laboratory work. Auditing will help for the rearrangement and further clarification of the procedures as well as operators according to the research and development.

Cost benefit of cell culture

The major limitation of cell culture is the expenditure of effort and materials that goes into the production of a relatively little tissue. The cost of producing cells in culture is about ten times than that of using animal tissue. To treat with KCC for a patient with 60% burn will cost approximately US$ 50,000.

Transport and storage temperatures

The storage of cell line needed liquid Nitrogen. That also very much higher for developing countries. Transportation can be done either -40⁰C or -20⁰C. Duration of storage will be 12 hours and long time respectively.

Culture techniques must be carried out under aseptic conditions, because animal cella grow much less rapidly than many common contaminants such as bacteria, mold and yest. Unlike microorganisms, cells from multi cellular animals do not exist in isolation and consequently are not able to sustain independent existence without the provision of a complex environment, stimulating blood plasma or interstitial fluid. This implies a level of skill and understanding to appreciate the requirements of the system and to diagnose problems as they arise. Tissue culture should not be undertaken casually to run one or two experiments.

Application of radiation for cell culture

Mainly other than sterilization of medical devices and other pharmaceuticals following advantages also can be taken from the gamma radiation.

	Production of cell lines by arresting their proliferation. This application will expose with the dose range of 20 to 60 gray of radiation exposure.
	Slight treatment of radiation for glass or polystyrene surfaces prior to start culturing, enhance the cell adhesion and its increase the cell proliferation (mainly due to slight negative charge (container provide the substrate ), extra cellular matrix proteins and proteglycans by the cell).

Environmental control

Control of physico-chemical environment is very important. Main factors are pH, temperature, osmotic pressure, Oxygen, Carbondioxide and tension should control preciously the physiological conditions " cell physiology has to be constant". Chemical batch variations such as medium supplements like serum and control of undefined elements like hormones and other regulatory substances and making replacements with define constituents, more practical. Tissue samples are invariably heterogeneous. Replicates even one tissue varies in their constituent cell types. After one or two passages, cultured cell line assume a homogeneous. Randomly mixing at each transfer and selective pressure of the culture conditions tend to produce homogeneous culture of the most vigorous type. Sub-culture each replicate sample will be identical and the characteristics of the line may be perpetuated over several generations or indefinitely if the cell line is stored in liquid N₂. Statistical variance has to be minimized.

Feeder cell line preparation

Dilution ratio of original cell line result different classes of fibroblasts. Bitamycin also can be used to grow pure DNA free cell line for the future KCC activities. Every three days KCC medium has to be changed for better environment for the cells. Now in the market, readymade purified keratinocyte mediums are available. In that medium we can grow keratinocyte cell layers without feeder cells. Moving of the KC culture flasks enhance the continuous spreading.

Fixation of KCC specimen for photography.

	Add 2-3 ml of triptopan blue (not much sterility is important).
	Medium takes away from the flask.(add 50ml of PBS and washing)
	Add 5ml of PBS and close the lid and shake the culture flask.
	Keep the specimen in -1500C and after a while add 50ml of 95% ethyl alcohol.
	Close the flask and take out and leave 30min in the room temperature.
	Keep some time under the laminar flow for the establishment.

Tips and tricks for successful cell culture laboratory.

	Transport medium antibiotic concentration and washing solution must be 3X cell culture medium concentration.
	Positive pressure laboratories can be benefited many advantages.
	Using out of range chemicals result toxic effect to the cells.
	Do not add any antimicotics for the cell culture medium other than recommended micotic such as AMPHOTERECIN B. (Micotics has a direct effect on cell wall)
	Every three months we must have a feeder cell line for the cell stock.
	Genetic lifetime of a cell will have effects on transplant cell layers.
	Single supplier recommended for the supply of chemicals and instruments.
	Cell culture laboratories only restricted for the cell culture work.
	Walls of the laboratory - gypsum laminated is the best.
	To filter the cell suspensions, cell sieves can be used.

List of References

	Cell and tissue culture laboratory procedures, A Doyle, Griffiths et al.
	Culture of animal cells, A manual of basic technique, R. Ian Freshney

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