Established cell lines (passage level >70) have been produced from a num ber of different types of hum an brain tum ours including m alignant astrocytomas and oligodendrogliom as (Ponten &
M acintyre, 1968; Giard et al, 1973; W esterm ark et al, 1973; Bigner et al, 1981a; Collins, 1983;
Rutka et al, 1987c). Most of these have been established from adult m alignant astrocytomas and
include some of these m ost w idely used for in vitro studies (e.g., U138MG, U251MG, U373MG
am ong others). However, there has been a m arked lack of established cell lines from m alignant astrocytom as from children and benign astrocytom as from both children and adults. In fact, th ere is only one established cell line d erived from an 8-year old bo y w ith a frontal
glioblastoma, SF-188 (Rutka et al, 1987c) b u t none from the lower tum our grades.
Established cell lines from m edulloblastom a are uncom m on (Batzdorf & Gold, 1974;
Friedm an et al, 1985; Jacobsen et al, 1985; Rutka et al, 1987a; Friedm an et al, 1988a; Tam ura et al,
1989a; Bigner et al, 1990a; Darling, 1990; Oakes et al, 1990; Pietsch et al, 1994). Initial studies in
the grow th of m edulloblastom a in vitro have concentrated on the description of short-term
cultures from these tum ours as either m onolayers, w here satellite colony form ation w as observed (Batzdorf & Gold, 1974), or in organ culture w here astrocytic differentiation was pro d u ced (H erm an & Rubinstein, 1984). The description of w ell characterised lines derived from meduUoblastomas have been pubUshed and are sum m arised in Table 1.
Cell line Origin G row th in
vitro
Reference
D283 Med Biopsy-abdominal metastasis Suspension Friedm an et al, 1985
D341 Med Biopsy-cerebeUum Suspension Friedm an et al, 1988a
D384 M ed Biopsy-cerebeUum Suspension Bigner et al, 1990a
D425 M ed Biopsy-cerebeUum Suspension Bigner et al, 1990a
D458 Med CSF-recurrent D425 Med Suspension H e et al, 1991
DAOY Biopsy-cerebeUum A dherent Jacobsen et al, 1985
ONS-76 Biopsy-cerebeUar tum our A dherent Tam ura et al, 1989a
ONS-81 Biopsy-cerebeUar tum our A dherent Tam ura et al, 1989a
MHH-MED- 1,2, 3 and 4
The m ost im p o rta n t difference b etw een the five cell lines established at D uke U niversity and those established by others is that the cell lines grow in suspension as loose aggregates of floating cells or larger aggregates resem bling m ulticellular tu m our spheroids
(Friedm an et al, 1985, 1988a, Bigner et al, 1990a, He et al, 1991). In com parison, Daoy, ONS-76
and ONS-81 grow as adherent monolayers (Jacobsen et al, 1985; Tam ura et al, 1989a). D283 M ed
w as established from a m ixture of digested tissue fragm ents and ascites cells. D341 M ed was p ro d u ce d from a m echanically disaggregated single cell suspension. A d h erent cells only persisted to passage level 3 b u t the non-adherent cells continued to grow. Both D384 M ed and D425 M ed w ere established from m aterial rem oved using a C avitron U ltrasonic Surgical A spirator (CUBA) and p u t into culture w ithout further digestion. Daoy, ONS-76 and ONS-81 were from mechanically disaggregated tum our biopsies w ithout enzymatic digestion.
Recently, Fults et al (1992b) established a cell Une, PFSK from a PNET in the cerebral
hem isphere of a 22 m onth old boy. To date this is the only example of an established PNET cell line derived from the cerebral hemisphere. Initially, cells proliferated as a m onolayer b u t after 10 days in culture, foci of cells were grow ing as m ultilayered aggregates on the m onolayer surface. These foci w ere then picked and transferred to m icrotitre w ells w hich pro d u ced sublines; PFSK-1 and PSFK-2C. The PFSK-2C cells were passaged independently giving rise to
the PFSK-2 subline w hich grows in clum ps w ith poor surface adherence. Recently, Pietsch et al
(1994) has established a further five cell lines from hum an PNETs, three were generated from cerebellar MB (MHH-MED-2, MHH-MED-3 and MHH-MED-4) and one from a PNET located in the spinal cord (MHH-MED-5) and another established from cells obtained from CSF of a patient w ith a recurrent MB (MHH-MED-1). All biopsies w ere m inced and placed into culture and adherent cells rem oved by two consecutive steps of plastic adherence for 90 mins. The four
MB cell lines grew in suspension as floating aggregates or as slightly adherent cells (Pietsch et
al, 1994). One of the earliest described m edulloblastom a cell line, TE671 (Me AUister et al, 1977),
has subsequently been show n to be identical to the RD cell line, originally derived from a
rhabdom yosarcom a (Stratton et al, 1989b).
Only tw o established cell lines have been described from ependym om as. One is NU2-
26 (N akagaw a et al, 1983) derived from a cerebellar ependym om a and the other, KM-SII
(Mihara, 1986) was established from a recurrent ependym om a developed in the 4th ventricle of a 4 year old boy. Both cell lines were produced by m echanical disaggregation and treatm ent w ith trypsin and grew as adherent cultures.
M orphology
The m orphological characteristics of cells derived from h u m an b rain tum ours have been review ed over the years (Lumsden, 1971; U nterham scheidt, 1972). Low grade astrocytom as have been show n to produce slowly proliferating cells w ith m arked morphological evidence of astrocytic differentiation. These cells have long processes w hich form a loose netw ork. On the
o ther h an d , m alignant gliom as tend to produce m orphologically heterogeneous p rim ary cultures. M ore hom ogeneous m orphological patterns of appearance develop w ith subsequent
passaging (Bigner et al, 1981b, Yang et al, 1990). Some workers have grouped established cell
lines into four classes on the basis of their appearance after m ultiple passages. These groups are designated epithelial (polygonal shape w ith clear, sharp boundaries; "pavement-like"), glial (cell body and processes), fascicular and fibroblastic (spindle-shaped, bipolar or stellate and
m ultipolar) (Bigner et al, 1981b).
C u ltu re s d e riv e d from m a lig n a n t a stro c y to m as sh o w classical cytological characteristics of anaplasia, including mitoses, giant cells, nuclear overlapping, heterogeneity in
nuclear size and shape as well as evidence of cell piling (Giard et al, 1973; M aunoury, 1977;
Bigner et al, 1981b; Rutka et al, 1987c). In vitro, ependym om a cell cultures have an epithelial-like
m orphology and form ation of rosette-like patterns w hen density is high (Nakagawa et al, 1983;
M ihara, 1986). In these cultures there is little cytological evidence of malignancy.
M eduUoblastomas, on the other hand, grow relatively well in short-term culture and
cells m ay persist in vitro for m any m onths (Wilson et al, 1966). U nfortunately, w ith time, these
cultures tend to degenerate or adventitious cells dom inate (Lumsden, 1971). There have been n o com prehensive studies on the identification of these cells grow n from surgical biopsy
specimens. However, W aghe et al (1973) dem onstrated the presence of proliferating cells using
tritiated thym idine. In prim ary cultures, m orphological cell types include small spindle shaped
or triangular cells w ith round, oval nuclei and scant cytoplasm. A stu d y by M ackillop et al
(1985) observed that 3 MB cultures grew in m onolayer w ith the presence of floating aggregates. The cells from these cultures w ere elongated b u t lacked the long processes seen in the astrocytom as. The "Duke" series of m edulloblastom a cell lines all grew in suspension and fo rm e d agg reg ates sp o n tan eo u sly . T hree sub lin es of PFSK-1 w ere sh o w n to h av e "m ultinucleated cells w ith broad flat m orphology", w hereas PFSK-2 grew in clum ps and
adhered less well to plastic (Fults et al, 1992b). Three cell lines (MHH-MED-1, 2 and 3) grew as
cell aggregates in suspension, the cell line MHH-MED-4 w as slightly adherent and M HH- PNET-5 m ainly com prised of adherent cells w ith spindle-shaped or stellate m orphology (Pietsch et al, 1994).
Cell line kinetics
Bigner et al (1981a) dem onstrated that established glioma cell lines reached higher saturation
densities than norm al hum an glial cultures. The doubling times of short-term cultures derived
from m alignant astrocytom a are com parable to those of norm al ad u lt brain (Pertuiset et al,
1985) and it is apparent that there is a stabilisation in doubling times over the first five passages
in vitro (Pertuiset et al, 1985). Reports in the literature indicate that established cell lines derived from h u m an gliom as show w ide variation in their doubling tim es, betw een 24-264 hours (Pertuiset et al, 1985; Cook et al, 1987; Zupi et al, 1988).
The heterogeneous m orphological appearance of cultures at quite high passage levels, suggests that various cell types are still present in these cultures, indicating that a m echanism is in operation that regulates the grow th of these cells within a culture. Other studies have show n that small subpopulations of endothelial cells were incorporated stably into short-term cultures
of h u m an gliom a for m any passage levels (Guner et al, 1977). How ever, some m ixed cultures
have been show n to be stable in culture, therefore indicating that doubling times are sim ilar for different cell types.
P opulation doubling tim es for established m edulloblastom a cell lines fall into tw o groups. Daoy, D283 Med, D341 Med, ONS-76 and ONS-81 have doubling times betw een 19-53 hours, while cell lines D384 Med, D425 M ed and D485 M ed have doubling tim es in the region
of 58-83 hours (Tamura et al, 1989a; Bigner et al, 1990a; Oakes et al, 1990). Of the m ost recently
established MHH-MED series, doubling tim es ranged betw een 61-72 hours. The population doubling times for PFSK cell line is 30 hours for sublines 1 and 2C and 48 hours for subline 2, w hile the ependym om a cell lines, NU2-26 and KM-SII have rapid doubling tim es in the region of 24 hours.
Karyotypic analysis
Established glioma cell lines generally contain stemlines or m odal chromosome num bers in the
near-triploid region (Mark et al, 1974; Bigner et al, 1983). This seems to be relatively stable at
high passages although some changes do occur, including gains or losses of copies of entire norm al or abnorm al chrom osom es as w ell as the appearance of new chrom osom al m arkers (Rey et al, 1983; Key et al, 1989). M edulloblastom a cell lines from the "Duke" series are near diploid and often have structural abnorm ality of chromosome 17, including an isochromosome 17 (Bigner et al, 1990b).
Am plification of the c-myc gene in cell lines w as reported (Bigner et al, 1990a) b u t no
am plification of N-myc, EGER or gli w as detected. A n 150-fold increase in the amplification of
the N -m yc gene was observed in a new m edulloblastom a cell line, MTS (Wasson et al, 1990). It
appears that m edulloblastom a cell lines have a high frequency of c-myc amplification and this
m ay be a requisite for such tum ours to establish in vitro (Wasson ef al, 1990). In contrast, Daoy is
tetraploid and has been show n to have amplification of the erbBl gene (Wasson et al, 1990). The
PFSK-1 cell lines are hypotetraploid w ith m any chrom osomal aberrations, whereas the PFSK-2 has a pseu d o d ip lo id karyotype w ith translocations t ( l :l l) , t(3:10) and t(17:22). Trisom y of
chrom osom e 8 of the PFSK-2C cells was also noted (Fults et al, 1992b). Cytogenetic analysis of
the cell lines in the M H H series show ed near diploid stem line karyotypes w ith clonal structural abnorm alities. A partial trisom y of I q w as found in tw o lines (MHH-MED-1 and M HH-M ED-4) an d chrom osom al regions 16q24, H q24 an d 10q24 exhibited stru c tu ra l
Interestingly the cell lines MHH-MED-2, 3 and 4 h ad no detectable losses on chrom osome arm
17p. The MHH-MED-2 cell line show ed double m inutes and amplification of c-myc as w ell as
over-expression of c-myc mRNA and protein (Pietsch et al, 1994). One ependym om a cell line,
KM-Sll is hyperdiploid w ith a m odal num ber of 56 and the other, NU2-26 is near diploid w ith a chrom osom e m odal num ber of 46. In a cytogenetic analysis of cultures derived from four ependym om as w ith different degrees of m alignancy (1-lV), num erical changes w ere seen in chrom osomes 17 and 22 and structural changes were seen involving chromosomes 10,12, and X
(Rogatto et al, 1993).