Viable Hybrids between Adherent Cells:
Generation, Yield Improvement,
Somatic cell hybridization was discovered and
introduced by Barski et al.
(1960) and Sorieul and
Ephrussi (1961). This technique allows one to examine
the result of introducing various genomes in different
functional states and from different species into the same
cell. Hybrid cells have been widely used in
various fields (genetics, cell biology, tumour biology,
virology) and the most famous hybrids are
One important application of somatic cell
hybridization is chromosomal gene assignment.
Breeding analysis, which is effective for this purpose
in lower animals and plants, is too slow in mammals
(even in mice the generation time is about 3 months)
and is impossible in humans. Gene mapping techniques
based on somatic cell genetics have been central
to the study of human genetics. In 1968, only two
genes had been mapped to specific autosomes, and a
decade later this number had risen to 300, mostly using
human-rodent somatic cell hybrids. Such hybrids
present the advantage to retain only a few human
chromosomes and they are now currently used as
donor cells in irradiation and fusion gene transfer
(IFGT) experiments for constructing detailed genetic
maps (Walter and Goodfellow, 1993).
Cell fusion was also used to analyse how specialized
cells acquire and maintain their differentiation.
The activities of somatic cells can be divided into two
main categories: essential or ubiquitous functions that
are indispensable for cell survival and growth and "luxury" or differentiated functions. Essential functions
continue to be expressed in hybrids, whereas
differentiated functions are subject to different regulations
(expression, extinction, activation) depending
on the histogenetic nature of the parental cells that
have been fused (see examples in Cassio and Weiss,
1979; Hamon-Benais et al.
, 1994; Killary and Fournier,
1984; Mevel-Ninio and Weiss, 1981). Cell determination
is, however, not modified in hybrids, as
extinction or activation requires retention of the
chromosomes coding for the appropriate regulatory
Somatic cell hybridization has not only shown that
tissue-specific genes are regulated by trans
factors, but has provided strong evidence for the existence
of tumour suppressor genes (Anderson and
Stanbridge, 1993). Cell fusion experiments have also
demonstrated that cellular senescence is a dominant
active process and that several genes or genes pathways
are implicated in the senescence program (Goletz et al.
Spontaneous fusion of cells in culture occurs at a
very low frequency. To obtain hybrid cells, inactivated
Sendai virus or, more commonly, polyethylene glycol
(PEG), which was introduced by Pontecorvo (1976), is
used as the fusogen. Cell hybridization has also been
performed by electrofusion on filters (Ramos et al.
2002). The inital products of fusion contain within a
common cytoplasm two or more distinct nuclei
from one single parent (homokaryons) or from both
(heterokaryons). Only a very small proportion of these
polykaryons will progress to nuclear fusion and then
through mitosis. Moreover, the first divisions of the heterokaryons and of their daughter cells often fail
because of abnormalities of the mitotic spindle and
abnormal chromosome movements. The formation of
viable hybrids from heterokaryons is thus a rare event,
and the use of selective methods that favour the survival
of the hybrids at the expense of the parental cells
is often a requisite. These selective methods are also
necessary because hybrid cells often grow more slowly
than parental cells and are rapidly overgrown by
A. Selective Methods
The best known of such methods is the application
of hypoxanthine + aminopterin + thymidine (HAT)
selection (Littlefield, 1964) for the fusion of cells deficient
in hypoxanthine guanosine phosphoryl transferase
) with cells deficient in thymidine
), but different combinations of selectable
markers can be used (Hooper, 1985), provided that the
two selective systems do not interfere. If the lines that
are fused have no selective markers, a good strategy is
to select sequentially for HGPRT deficiency (thioguanine
resistance) and ouabain resistance in one parental
cell line. Then this marked cell line may be fused
with any unmarked cell line and hybrids selected in
HAT + ouabain (Jha and Ozer, 1976). Other couples
of selective markers, such as TK deficiency (5-bromo-
2'deoxyuridine resistance) and neomycine resistance,
can also be used. For producing primate-rodent
hybrids, the selection of an HGPRT- rodent parent is
sufficient because rodent cells are more resistant to
ouabain than primate cells. Moreover, hybrid cells can
also be isolated on the basis of their size, morphology,
growth parameters, and DNA content.
B. Yield of Viable Hybrids
Whatever the method used to isolate hybrids, the
most important is to optimize the fusion conditions in
order to obtain a number of viable hybrids as high as
possible. In the best cases the fusion of several millions
of parental cells leads to the formation of only a few
hundred hybrids and often the yield of viable hybrids
is much lower, as illustrated in Table I for hepatomaderived
hybrids. The protocol described here has been
used routinely to produce large amounts of hybrid
clones between differentiated rat hepatoma cells and
various cells of different histogenetic origin and of different
species, particularly mouse and human fibroblasts
(Mevel-Ninio and Weiss, 1981; Sellem et al.
Moreover, some of the hybrids obtained have been
used themselves as partners of fusion and new hybrids
were generated successfully using exactly the same
method (Bender et al.
, 1999; Hamon-Benais et al.
The most important parameters in fusion experiments
are the yield of viable and growing hybrids and their
stability. Thus it is recommended to define optimal
fusion conditions and to vary different parameters,
particularly the ratio of parental cells, for improving
the yield. It is also recommended to analyze regularly
hybrid clones for their phenotype and chromosomal
PEG 1000 ultrapure (Merck, Cat. No. 9729)
Trypsin (pig pancreas; United States Bioch. Corp.,
Cleveland, OH, Cat. No. 22715)
Complete growth medium (available from local
Serum-free growth medium (available from local
Selective complete growth medium (available from
35- and 50-mm tissue culture dishes (Falcon, Cat. No.
15-ml tube (Falcon, Cat. No. 352099)
22 × 22-mm sterile glass coverslips
A. Before Fusion
- 50% PEG (for fusion): Autoclave PEG 1000. This
both liquifies and sterilizes the PEG. Cool it to 50°C and mix with an equal volume of sterile serum-free
medium prewarmed for a short period at 50°C. Adjust,
if necessary, to the desired pH with 1.0 M NaOH (range
of pH generally used is 7.2-7.9). This solution can be
stored at 4°C for up to 2 weeks.
- 0.25 and 0.05% trypsin (to detach fusion products):
To make 100ml of solution, solubilize 0.8g of NaCl,
0.04g of KCl, 0.058g of NaHCO3, 0.1g dextrose, and
0.25g (0.25%) or 0.05g (0.05%) of trypsin. Complete to
100ml distilled water. Incubate at 37°C for 1-2h. Sterilize
by filtration and store at 4°C (rapid use) or -20°C.
Grow parental cells in nonselective medium
- Inoculate the mixture of parental cells to be fused
into several 50mm tissue culture dishes containing
complete (nonselective) growth medium. The total
number of cells has to be adjusted to occupy all the
dish surface, such that the fusion will be done on cells
that are in close contact. For 50mm petri dishes, the
total cell number could vary from 5 × 105 to 4 × 106 depending on the density at confluence of the cell
lines used. Although equal numbers of parental cells
are generally recommended, use different ratios of
parental cells (see Table II and Section IV).
- Incubate the mixed cultures a few hours (4h to
overnight). This allows the cells to adhere to the
support and to establish contacts with neighbors.
- Warm the serum-free medium and the 50% PEG
solution to 37°C.
- Remove the medium thoroughly from the
culture and wash once with 5ml of serum-free
- Add gently 3.0ml of PEG 50% all over the cell
- After 45 s aspirate the PEG.
- Exactly I min after PEG treatment, add 5ml of
- Aspirate half the medium and add 2.5ml of
- Repeat step 8 four times.
- Aspirate all the medium.
- Add 5ml of complete growth medium and let
the cells recover for at least 2h and no more than 12h.
: Steps 4 to 11 must be done dish per dish
control dishes must be included. They will be treated as the others except that the PEG solution will be
replaced by serum-free medium.
C. After Fusion
- Aspirate the medium and add 3ml of 0.25%
trypsin per dish.
- As soon as the cell layer begins to detach, add
3 ml of 0.05% trypsin and detach the cells by repeated
- Add the cell suspension in a 15-ml tube containing
2ml of complete growth medium (to arrest the
trypsin action). If necessary, rinse the dish with 2ml
medium and add this medium to the tube containing
- Centrifuge the cells at 500g (1500rpm) for 5min
at room temperature.
- Resuspend thoroughly the cell pellet in complete
growth medium and count the cell number in a hemacytometer.
Using the described protocol, generally at
least 80% of the cells are recovered after fusion.
- Pool, if necessary, cells recovered from identical
dishes and inoculate different numbers of cells
(103-106) in either culture dishes or on 22-mm glass
coverslips (in 35-mm dishes).
- Incubate the cells at least overnight (eventually a
few days) in complete growth medium before adding
selective medium that will kill the parental cells and
let the hybrid cells survive.
- At regular intervals, watch for the appearance of
growing hybrid colonies. Count their number in dishes
or coverslips that contain well-isolated colonies. From
this number the yield of growing hybrids can be calculated.
For one fusion, this yield is equal to the total
number of hybrid clones obtained divided by the total
cell number of the minority parent engaged in the
- Use dishes that contain a small number of
colonies to isolate independent hybrid clones (one per
dish will be scraped, subcultured, characterized, as
soon as possible, frozen, and recharacterized). From
dishes that contain a lot of colonies, if this situation
arises, hybrid cell populations can be obtained in mass
and studied rapidly.
- Use glass coverslips to control by cytogenetic
methods the hybrid nature of the clones and to test if
their chromosomal content is stable with time in
culture. The karyotyping in situ method, described by
Worton and Duff (1979), is highly recommended. This
method is easy, of general application for adherent
cells, and can be performed on small colonies even a
few generations after fusion (Fig. 1). Glass coverslips can also be used for phenotypic characterization of
|FIGURE 1 Karyotypic analysis of emerging hybrids. The in situ
karyotyping method (Worton and Duff, 1979) was performed on rat
hepatoma-derived hybrid colonies 8 days after fusion. The colony
shown was composed of 17 cells, 1 of which was in metaphase.
This metaphase contains 100 chromosomes, corresponding to the
expected sum of the mean chromosome number of each parent (46
and 52, respectively).
The production of hybrid clones in large amounts
depends greatly on the parental cells (cell type and
growth capacity) and on the fusion conditions. These
two points are illustrated in Tables I and II for rat
hepatoma-derived hybrids. The frequency of occurrence
of hybrids between rat hepatoma and normal
fibroblast was particularly low compared to other
hepatoma-derived hybrids (Table I). Consequently,
various fusion conditions were tested. The use of
unbalanced ratios of parental cells is one of the most
important parameters to improve the hybrid yield
(Table II). Therefore, to save time and to obtain the
highest number of hybrid cells, it is recommended to
fuse parental cells in different ratios.
Mixed parental cell populations that have not been
treated by PEG can give rise to colonies that grow in selective medium at low frequency. These colonies
could be either spontaneous hybrids or revertants
from parental cells. The isolation of revertants is one
of the most common difficulties that may arise in
selecting hybrids. Therefore the hybrid nature of the
cells selected has to be verified by checking their chromosomal
- Some PEG preparations produce enormous
lethality, whereas ultrapure PEG from Merck results in
acceptable levels of lethality (generally below 20%).
- Detach and dissociate very carefully cells after
fusion (avoid aggregates).
- Because it is impossible to predict if hybrid
clones will be produced with a high yield or not, once
fusion has been performed and the products of fusion
detached, inoculate them at different concentrations
(that could cover a 1000× range) such that well-isolated
hybrids could be obtained even if the yield varies from
10-6 to 10-3.
- A lower number of hybrid clones is obtained if
the cells are not detached after fusion.
- A lower number of hybrid clones is obtained if
the mixture of parental cells is fused in suspension.
This result is due to the fact that the relative proportion
of binucleate heterokaryons is higher in monolayer
fusion, whereas suspension fusion favours the
formation of giant heterokaryons that die soon after
fusion. Hence, there is no reason and no advantage
to fuse adherent cells in suspension as often
recommended (except if one of the parent grows in
I thank M. C. Weiss for training in cell culture and
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