Algoritmos heurísticos

P reparation of disaggregated surgical specim ens

Biopsy sam ples taken at surgery were im m ediately placed in collection m edium (CM) and tran sp o rted to the laboratory. In m ost cases, the sam ples w ere received w ithin 1-4 h ours of

rem oval. The biopsy samples were transferred to a plastic petri dish, together w ith some fresh biopsy collection m edium and any non-tum our m aterial was rem oved by dissection. The tissue fragm ents w ere then w eighed and chopped into l-2m m pieces using crossed scalpels. The blades were changed betw een biopsies to ensure that sharpness was m aintained and the biopsy w as cleanly cut rather than mangled. The fragm ents w ere then transferred to a 30ml universal container and resuspended in com plete grow th m edium (GM) (H am 's F-10, buffered w ith 25mM HEPES and supplem ented w ith 10% v / v selected foetal calf serum and 100 u n its /m l penicillin and lOOmg/ml streptom ycin). A volum e of 1ml crude collagenase solution was

ad d ed to 2ml of complete GM, to give a final working concentration of = 666 un its/m l.

The tissue was incubated for betw een 1-4 hours at 37°C. W hen the tissue was reduced to sm all aggregates rather than a single cell suspension, a further 7mls of com plete GM was ad d ed and the preparation agitated several times. This tissue suspension w as then centrifuged for 5 m inutes at 1000 rpm . The supernatant w as discarded and cells w ere resuspended in fresh com plete GM and plated into a small (25cm^) flask. The flask w as incubated at 37°C overnight, after w hich the m edium and the non-adherent m aterial w ere rem oved. The culture w as re-fed w ith fresh m edium and examined twice a week and re-fed as required.

P reparation of explant culture for sm all surgical biopsies

This technique was developed for small biopsy samples taken during stereotactic neurosurgical operations (Thomas et al, 1984). This procedure w as also carried out if the tum our specim en w as less th an 5m m in size. The biopsy sam ples w ere collected as m entioned previously, w eighed and w ashed w ith GM. Using a sterile 1ml pipette, two small spots of silicone grease (Edw ards high vacuum grease) were placed approxim ately 1cm apart on the base of a 25cm^ plastic cell culture flask. Using sterile forceps, the fragm ent w as placed betw een the spots of silicone grease in a drop of biopsy collection m edium . A sterile glass coverslip (9 x 22mm, N o.l thickness. M arathon Laboratory Supplies, London) was placed over the biopsy and spots of silicone grease. The coverslip was pressed dow n using the sterile forceps un til the biopsy sam ple w as held securely dow n against the plastic surface and the coverslip w as firm ly anchored by the silicone grease in such a w ay that the biopsy w as half u n der the coverslip and h alf out of it, so that the exposed half of the tum our biopsy w as in direct contact w ith the m edium . Five m l of complete GM was added carefully dow n the side of the flask, taking care n ot to dislodge the coverslip or explant. The flask w as then incubated at 37°C. W hen significant cell grow th from the biopsy w as evident, the coverslip and the rem ains of the biopsy were rem oved w ith sterile forceps and the culture refed w ith fresh complete GM.

P reparation of tum ours for grow th as floating aggregate cultures

Preparation of plasticware

In order to prevent adherence of cells to the base of the tissue culture treated flasks. N oble or Bacto agar solutions (10% w /v ) were m ade w hen required and diluted in prew arm ed GM to

give a 0.5% w / v solution. This solution was im m ediately used to coat the base of 25cm^ flasks (5ml/flask). The flasks were allowed to stand for about 30 m inutes so that the agar solidified.

Maintenance of floating aggregate cultures

T um our biopsies w ere collected and disaggregated as described previously. G enerally only samples that w ere thought to be m edulloblastom as w ere additionally p repared in this way. If sufficient m aterial w as received, 5mls of cell suspension (containing approxim ately 5 x 10^ cells) in GM w as introduced to a 25cm^ agar-based flask. After 2-3 days in culture, the GM and

floating aggregates were rem oved, using a 10m l pipette, and transferred into a new agar-coated

flask. This was a necessary procedure since cells w ere still able to m igrate u n der the agar to the base of the flask w here cells w ould grow as a m onolayer, even if detachm ent of the agar did not occur. O n re-feeding, the spent GM and the floating aggregates w ere transferred to a 30ml universal container and w ere then allow ed to settle u n d er gravity. The spent m edium was carefully rem oved (care being taken not to lose any floating aggregates) and new m edium introduced, after which the entire contents w ere transferred into a new flask.

R e-feeding

C ultures w ere re-fed on the first day after plating follow ing enzym atic disaggregation, to remove any d ead cells, residucil enzymes or red blood cells. Refeeding w as perform ed every 2-3 days, or w hen the p H changed from neutral to acidic. Slow growing cultures w ere fed at either weekly or fortnightly intervals. The process of re-feeding involved rem oving the old m edium and replacing it w ith an equal volum e of fresh complete GM. Generally, small (25cm^) and m edium (75cm^) flasks required 5ml and 10-20ml of m edium respectively.

Passaging cultures (subculturing)

Generally, cultures w ere passaged once they reached confluency. For prim ary cultures, this varied from betw een 2-5 weeks. Explant cultures, however, did not reach confluency as such and w o u ld rath e r grow as islands of heaped, non-contact inhibited cells and th u s w ere passaged prior to entering the plateau phase of growth.

The depleted m edium was aspirated and the cells w ere rinsed twice w ith lOmls of HBSS. The m onolayer w as then rinsed w ith 1ml of trypsin solution (0.25% trypsin, Gibco BRL). A further aliquot of 3ml trypsin was added to the flask, w hich was then incubated for betw een 10-15 m inutes, or until such time that the cells detached from the plastic. In some instances, scraping of cells w ith a rubber cell scraper was needed w here a sub-population of cells h ad to be rem oved by mechanical m eans since they w ere still strongly attached to the base of the flask. The cell scraper was introduced into the flask and the adherent ceUs gently scraped off the base of the flask. This enabled the harvesting of the majority of cells present in the flask. Seven m l of com plete GM w as added to the flask and the cell suspension transferred to a sterile universal container and centrifuged for 5 m inutes at 1000 rpm . The supernatant w as discarded and the

cells resuspended in 10ml of fresh complete GM. The cells w ere counted and either p repared for cell freezing or further passage. The total contents of the prim ary cultures w ere passaged from a 25cm^ flask into a 75cm^ flask (i.e., 1: 3 split).

Cell counting using a ZM C oulter counter

W hen the cells w ere resuspended w ith fresh complete GM, following centrifugation, 0.4ml of the cell suspension was pipetted into a pot containing 19.6ml Isoton II. This w as counted using a ZM Coulter Counter, giving a reading, which was then m ultiplied by 1000, to give the actual

total cell count in the 10m l suspension.

Cell freezing

The procedure w as the same as that for passaging cells, to the point w here a cell count w as carried out, after which cells were recentrifuged to rem ove the supernatant. The cell pellet was then gently resuspended in a 10% solution of DMSO in com plete GM, at a density of 1 x 10^ cells/m l. The DMSO acted as a cryoprotectant. One m illilitre aliquots containing 1 x 10^ cells were transferred into a freezing vial (cryotubes. Nunc). The vial was sealed and placed either in

a polystyrene box which was placed in a -70°C freezer for a m inim um of 8 hours before being

transferred to the liquid phase (-196°C) of liquid nitrogen freezer, or in a program m able controlled rate freezer (Planer Biomedical Ltd) set to produce a cooling rate of 1°C a m inute, for optim um preservation. Cultures were frozen dow n routinely betw een passage level 2 and 3.

To revive the cells from the liquid nitrogen bank, the vial was rem oved from the liquid phase and quickly transferred to a covered plastic container w ith w ater at 37°C (a precaution to avoid an explosion caused by any liquid nitrogen trapped w ithin an incom pletely sealed vial). Once thaw ed, the contents were rem oved from the vial and then slowly diluted into lOmls of complete GM in a 75cm^ flask and incubated overnight. The following day the ceUs w ere re-fed.

P opulation doubling tim es and saturation cell densities

The population doubling tim e (PDT) is defined as the tim e taken for the cell p o pulation to increase 2-fold during exponential growth. Population doubling tim es w ere calculated for a sm all num ber of short-term cultures from childhood brain tum ours. Cells w ere seeded into thirty 25cm^ culture flasks at an initial density of 5 x 10^ cells/m l in 5mls com plete GM p er flask. The flasks w ere incubated at 37°C u n d er standard conditions. A t each tim e point, two flasks w ere rem oved from the incubator, the m onolayers w ashed w ith HBSS and the cells detached w ith trypsin, centrifuged and resuspended in HBSS. The cells w ere counted using a ZM C oulter Counter and the num ber of cells/cm ^ w as calculated. PDTs w ere calculated from

Fig 4: Derivation of doubling