133

Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946,
DOI 10.1007/978-1-62703-128-8_9, Springer Science+Business Media, LLC 2013
Chapter 9
Simultaneous Cloning and Selection of Hybridomas
and Transfected Cell Lines in Semisolid Media
Bert Wognum and Tracy Lee
Abstract
Selection and cloning are essential but often laborious and time-consuming steps during the generation
of hybridomas and genetically modi ed cell lines that produce monoclonal antibodies or other proteins
with desired properties. Methods for the simultaneous selection and cloning of hybridomas and trans-
fected cell lines (e.g., CHO-S cells) in semisolid methylcellulose-based media have been developed.
By using semisolid selection media, the cells that survive the selection process proliferate and form colonies
of cells that remain physically separated from other colonies. Each colony thus originates from a single
hybridoma or transfected cell and can be isolated and characterized separately. This approach avoids the
isolation of multiple identical clones and the loss of useful clones due to overgrowth by other faster-
growing, but possibly nonproducing clones, which are major problems of conventional procedures in
liquid media. In this chapter, protocols are described for the generation of mouse hybridomas by fusion of
spleen cells from immunized mice with myeloma cells and the subsequent selection and cloning of hybri-
domas in semisolid selection media. Protocol are also described for selection and cloning of transfected cell
lines using semisolid antibiotic-containing selection media, as well as strategies to optimize selection and
cloning in serum-containing, serum-free, and chemically de ned selection media.
Key words: Monoclonal antibodies , Methylcellulose , Colonies , Transfection , Chinese Hamster
Ovary Cell Line

Procedures for hybridoma or transfected cell line generation are
aimed at the development of stable clonal cell lines that produce
high levels of monoclonal antibodies or other proteins with desired
properties. Conventional methods for cell line development involve
the selection, screening and cloning of hundreds or even thousands
of cell cultures to generate stable clonal cell lines. Traditionally
1. Introduction
134 B. Wognum and T. Lee
the selection and cloning of newly generated cell lines is done by
culturing the cells in multiple parallel cultures (e.g., in individual
wells of 96-well plates), screening each culture for the presence of
the protein of interest and then cloning the selected cell line by at
least one round of culturing under limiting dilution conditions,
and then screening each culture for the presence of cells that
produce the desired antibody or recombinant protein. These
methods require multiple culture and screening steps.
This chapter describes methods for simultaneous cloning and
selection of hybridomas and transfected cell lines using semisolid
media. By plating the cells in a semisolid selection medium after
fusion or transfection the progeny of the selected cells stay
together and form distinct colonies that can be harvested and
screened individually. As each colony is derived from a single
cell, selection of replicate clones is avoided. In addition, smaller,
slow-growing clones remain physically separated from larger
faster-growing clones and can be isolated and screened separately,
thus avoiding loss of the smaller clones due to overgrowth by
larger, faster growing clones and therefore increasing the diver-
sity of clones that can be identi ed and isolated. This approach
can signi cantly reduce the time and work needed to generate
stable clonal cell lines that express the protein of interest.
The high viscosity of semisolid media used for selection and
cloning is achieved by the addition of methylcellulose to the
culture medium. Methylcellulose is a relatively inert polymer with
good optical clarity and suitable viscosity at concentrations of
~0.91.5%. It is possible to make methylcellulose-based selection
and cloning media in ones own lab from individual components,
but prescreening of methylcellulose batches, fetal bovine sera,
cytokines, and other components is required to avoid large
batch-to-batch variability in performance. Alternatively commer-
cially available media can be used that have been developed
speci cally for hybridoma development or transfected cell line
generation. Suggestions for making and testing semisolid media
from liquid media and culture-tested concentrated methylcellulose
solutions are also provided.

1. Biosafety cabinet certi ed for level II handling of biological
materials.
2. Incubator with humidity and gas control to maintain 37C,
>95% humidity, and 5% CO
2
.
3. Inverted microscope.
2. Materials
2.1. Laboratory
Equipment
and Supplies
135 9 Cloning in Semi-solid Media
4. Low speed bench centrifuge.
5. Liquid nitrogen tank and freezing head; optional.
6. Freezing container (i.e., Mr. Frosty Nalgene Catalog #5100);
optional.
7. Culture supplies: 15 and 50 mL conical polypropylene centri-
fuge tubes, 1, 5, and 10 mL sterile pipettes, 10 cm petri dishes,
3 and 12 mL syringes; 16-gauge blunt end hypodermic
needles, 96- and 24-well tissue culture plates, tissue culture
asks.
8. Automated cell counter or hemocytometer and routine light
microscope, 0.4% trypan blue dye solution for viable cell counts.
1. Myeloma Cell Line. The myeloma cells should be mycoplasma
free, fuse well and allow the formation of stable hybridomas
that continually secrete speci c monoclonal antibodies. Parental
myeloma cells that meet these criteria (such as SP2/0 and
X63Ag8.653) are widely available. Whenever possible, obtain
a parental myeloma cell that has been proven to yield stable
hybridomas.
2. Immunized mouse 14 days after nal antigen boost.
3. Sterile sets of ne, sharp scissors and forceps for animal dissec-
tion. Sterilize by autoclaving for 40 min at 121C.
4. ClonaCell-HY monoclonal antibody development kit
(STEMCELL Technologies, Catalog #03800). The kit contains
Medium AClonaCell-HY perfusion medium and hybridoma
expansion medium, 500 mL.
Medium BClonaCell-HY fusion medium, 500 mL.
Medium CClonaCell-HY Hybridoma Recovery medium,
100 mL.
Medium DClonaCell-HY Hybridoma Selection medium
containing hypoxanthine, aminopterin, and thymidine
(HAT), 90 mL.
Medium EClonaCell-HY Hybridoma growth medium
containing hypoxanthine and thymidine (HT), 500 mL.
Polyethylene GlycolClonaCell-HY PEG solution, pretested
for cell fusion, 1.5 mL.
Store according to suppliers instructions.
5. Additional supplies for detecting hybridoma antigen-speci c
antibody production (e.g., using ELISA, immunocytochemis-
try, immunoblotting or ow cytometry).
1. A cell line capable of growing in suspension under nonadherent
conditions, e.g., CHO-S (available from ATCC; www.atcc.org ).
For examples of cell lines that have been found suitable for
2.2. Generation
of Mouse Hybridoma
2.3. Transfection,
Selection and Cloning
of Cell Lines
136 B. Wognum and T. Lee
selection and cloning in methylcellulose-based media (see
Note 1).
2. A liquid medium suitable for expanding the cell line prior to
transfection, maintaining the cells during the recovery phase
immediately after transfection before plating in semisolid
medium and for expanding cloned cell lines after selection and
cloning, e.g., ClonaCell-CHO CD Liquid (catalog #03817,
STEMCELL), for CHO-S and other CHO sublines that have
been adapted to suspension cultures (see Note 2).
3. An appropriate DNA vector containing the gene of interest
and a selectable marker gene, encoding, e.g., for resistance to
neomycin, hygromycin-B, or other antibiotics.
4. Antibiotic appropriate for the selectable marker gene used,
e.g., G418, Hygromycin B.
5. Reagents and/or equipment for DNA transfection of eukary-
otic cells.
6. ClonaCell-TCS, methylcellulose-based semisolid medium for
transfected cell selection (catalog # 03814, STEMCELL).
7. ClonaCell-CHO-CD chemically de ned protein-free medium
(Catalog # 03815, STEMCELL) or ClonaCell-CHO-ACF
Medium (Catalog # 03816, STEMCELL) for selection and
cloning of CHO cells in the absence of serum.
8. A methylcellulose stock solution, e.g., 2.6% methylcellulose in
Iscovess MDM (Catalog # 04100, STEMCELL) for prepar-
ing a semisolid selection medium with a liquid medium of
choice (see Notes 24).

An overview of the protocol is shown in Fig. 1 .
1. Thaw the parental myeloma cells and culture in ClonaCell-HY
Pre-Fusion Medium (Medium A) for at least 1 week prior to
fusion to ensure that the cells are well adapted to ClonaCell-HY
medium. Seed cells at approximately 5 10
4
cells/mL and
passage every 2 days. The suggested maximum cell density is
approximately 4 10
5
cells/mL, although a cell density of up
to 8 10
5
cells/mL is acceptable.
2. Calculate the cell growth rate at every passage. The day before
the fusion, count the viable cells and split so that at least 2 10
7

parental myeloma cells will be available for fusion. The cells
should be at early-mid log phase growth prior to fusion.
3. Methods
3.1. Hybridoma
Development
3.1.1. Prepare Myeloma
Cell Line
137 9 Cloning in Semi-solid Media
The recommended cell density for fusion is 2 10
5
cells/mL.
Only 100 mL of these cells is needed, but 200 mL should be
cultured to ensure suf cient cell numbers for fusion.
3. Harvest the parental myeloma cells by centrifuging in a 50 mL
conical centrifuge tube at room temperature (RT) at 300 g
for 10 min. Wash three times by using 30 mL of ClonaCell-HY
Fusion Medium (Medium B). Remove the supernatant by
pipette and resuspend the cell pellet in 25 mL of Medium B
(see Note 5).
4. Count live cells using a viability stain (e.g., Trypan Blue). The
viability of parental myeloma cells should be >95%.
5. Calculate the volume of cell suspension that contains 2 10
7

viable cells. Keep cells at RT or 37C until fusion (up to ~3 h).
Fig. 1. Procedure for hybridoma selection and cloning in semisolid medium.

138 B. Wognum and T. Lee
1. Sacri ce an immunized mouse according to procedures
recommended by your institution and wash the fur with 95%
ethanol. Clip fur and pull back to expose chest. Remove spleen
and place in a sterile petri dish containing 5 mL of Medium A.
Trim off any large pieces of fatty tissue.
2. Disaggregate the spleen into a single cell suspension. Transfer
the spleen to a nylon or metal screen placed on top of a 50 mL
conical centrifuge tube, and use the plunger of a 3 mL syringe
to grind the cells out of the spleen. Rinse the screen with
Medium B to assist the cells through the screen. Only the
spleen membrane should remain on the screen. Gently pipette
the cells up and down to disrupt clumps.
3. Wash splenocytes three times in 30 mL of Medium B, centri-
fuging at 400 g (~1,350 rpm) at RT for 10 min each time and
removing the supernatant by pipette. After the nal wash resus-
pend the cells in 25 mL Medium B (see Note 5).
4. Count live cells using viability stain (e.g., Trypan Blue).
Calculate the volume of cell suspension that contains 1 10
8

cells. Keep cells at RT or 37C until fusion (up to ~3 h).
1. Prepare PEG and media (Medium A, B, C) for fusion by pre-
warming to 37C. If using fusion Method A, prepare a 37C
water bath.
2. Add 2 10
7
parental myeloma cells and 1 10
8
viable spleno-
cytes to a 50 mL conical centrifuge tube and centrifuge for
10 min at 400 g . Aspirate off supernatant taking care not to
disrupt cell pellet. Complete removal of the supernatant is
essential to avoid dilution of PEG in the next step.
3. Fuse cells using one of the two methods outlined below.
1. Disrupt the cell pellet by gently tapping the bottom of the
tube. The pellet must be disrupted for optimal fusion. Slowly
add 1 mL of ClonaCell-HY PEG Solution (PEG) to the pellet
dropwise using a 1 mL pipette, over a period of 1 min without
stirring. Continually stir the cells gently, with the pipette tip,
over the next minute.
2. Add 4 mL Medium B to the fusion mixture, continuously
stirring as before, over a period of 4 min.
3. Slowly add 10 mL Medium B to the cells. Incubate for 15 min
in water bath at 37C.
4. Slowly add 30 mL of Medium A and centrifuge the cells at
400 g for 7 min.
5. Discard the supernatant and wash cells with 40 mL of Medium
A to ensure that all PEG is removed.
3.1.2. Harvest Spleen
and Prepare Spleen Cells
3.1.3. Cell Fusion
Method A
139 9 Cloning in Semi-solid Media
6. Slowly resuspend the cell pellet in 10 mL of ClonaCell-HY
Hybridoma Recovery Medium (Medium C). Transfer the cell
suspension to a T-75 cm
2
tissue culture ask containing 20 mL
of Medium C (total culture volume = 30 mL). Incubate for
1624 h at 37C in 5% CO
2
.
1. Disrupt the cell pellet by gently tapping the bottom of the
tube. Add 0.5 mL of ClonaCell-HY PEG Solution (PEG)
dropwise to the pellet using a 1 mL pipette. Centrifuge
the mixture at 133 g at RT for 3 min. Aspirate off all PEG
(see Note 6).
2. Carefully add 5 mL of Medium B dropwise to the pellet while
gently swirling the tube to resuspend the cells.
3. Slowly add 5 mL of ClonaCell-HY Hybridoma Recovery
Medium (Medium C) to the solution. Continue to swirl the
tube.
4. Transfer the cell suspension to a T-75 cm
2
tissue culture ask
containing 20 mL of Medium C (total culture volume = 30 mL).
Incubate for 1624 h at 37C in 5% CO
2
. There will still be
clumps of cells at this point which will break up overnight. Be
gentle with these cells.
1. On the day of the fusion, place ClonaCell-HY Hybridoma
Selection Medium (Medium D) at 28C and thaw overnight.
On the day after the fusion, shake the bottle vigorously to mix
contents well and let warm to RT.
2. Transfer fused cell suspension into a 50 mL conical tube and
centrifuge for 10 min at 400 g at RT. Remove the superna-
tant. Resuspend the cells in Medium C to a total volume of
10 mL. It is critical not to exceed the 10 mL nal volume.
If you wish to add any additional cytokines or supplements
to Medium D, include this volume in the total 10 mL.
3. Transfer the 10 mL cell suspension into the 90 mL of Medium
D. Mix thoroughly by gently inverting the bottle several times.
Let sit for 15 min to allow the bubbles to rise to the top.
Using a 12 mL syringe and 16 gauge blunt-end needle,
aseptically plate out 9.5 mL of cell suspension medium into
each of ten 100 mm petri plates (see Note 7). Tilt each plate to
evenly distribute the medium to cover the bottom of the plate.
Avoid the introduction of bubbles during plating.
4. Incubate plates at 37C in 5% CO
2
(see Note 7). Do not dis-
turb plates for 1014 days.
1. 1014 days after cells are plated in Medium D, examine the
plates for the presence of colonies visible to the naked eye
(Fig. 2 ) (see Note 8). Remove isolated colonies from the plates
Method B
3.1.4. Selection and
Cloning of Hybridoma Cells
3.1.5. Screening
and Harvesting of Clones
140 B. Wognum and T. Lee
using a pipettor set to 10 L and sterile pipette tips. Pipette
each clone into an individual well of a 96-well tissue culture
plate containing 200 L of ClonaCell-HY Growth Medium
(Medium E). With the pipettor set at 150 L, pipette the entire
contents of the well up and down several times to resuspend
the cells. Ensure a new sterile tip is used for each colony to
maintain clonality of the colony.
2. Incubate the plates at 37C in 5% CO
2
for 14 days without
feeding. By the fourth day, most wells should have a high cell
density and medium that is turning yellow. As the colonies
have different growth rates, media in some wells may turn
yellow sooner than 4 days. It is a good idea to pick clones of
different sizes as slower growing clones (i.e., smaller colonies)
are often very good antibody producers.
3. Transfer 150 L of supernatant from each hybridoma to a
separate well on a new 96-well plate and analyze by using an
assay system appropriate for the antigen involved (e.g., ELISA,
ow cytometry, Western Blotting, etc.).
4. Add 150 L of fresh Medium E to every well of the original
hybridoma containing plates and return the plates to the
incubator.
Fig. 2. Colonies of hybridoma cells after 14 days of culture in ClonaCell-HY Medium D.

141 9 Cloning in Semi-solid Media
5. Gently resuspend the hybridomas that showed a positive
response in step 3. Transfer 100 L of cells to each of two wells
of a 24-well plate, containing 1 mL of Medium E.
6. When cells have grown to a suitable density (approximately
4 10
5
cells/mL), freeze the cells from one well and expand
the remaining positive clones in a T-25 cm
2
tissue culture ask
containing 5 mL of Medium A and 5 mL of Medium E. This
step adapts the cells to growth in Medium A. In addition, keep
a sample of cells in Medium E, in case the cells dont adapt well
to the 1:1 mixture. The cryopreserved cells serve as backup in
case the cultured cells are lost, or in case antibody expression is
lost and recloning of the cultured cells is unsuccessful.
7. When cells have grown to a suitable density (approximately
4 10
5
cells/mL), transfer 510 mL of cell culture by pipette
into 20 mL of Medium A in a T-75 cm
2
. Adjust the volume of
cells to ensure the nal cell concentration is between 1 10
4

and 5 10
4
cells/mL. Maintain expanded hybridomas in 100%
Medium A at a concentration of 5 10
4
5 10
5
cells/mL.
More aliquots of cells can be frozen at this point in order to
secure the supply of the hybridoma clone.
8. Recloning of the clone of interest may be performed if desired
(see Note 9).
An overview of the protocol is shown in Fig. 3 .
The following is a general procedure which may require addi-
tional modi cations and necessary controls based on the choice of
cell line, vector, transfection method, and other experimental
requirements. Although many nonadherent cell lines and some
adherent cell lines grow well in semisolid medium and form tight
colonies, some do not. Cell lines that have been demonstrated to
grow in semisolid media are listed (see Note 1). For cell lines that
have not been tested before using this procedure the cloning
ef ciency and growth properties (speci cally, colony size, morphol-
ogy, cell viability after colony harvesting) should be determined
prior to using semisolid medium for selection and cloning after
transfection or for subcloning established cell lines. Since antibi-
otic resistance of a given cell line is dependent on the culture con-
ditions and may vary over time with continued passaging or
subcloning, it may be necessary to assess the minimum lethal anti-
biotic concentration in semisolid medium prior to transfection (see
Note 10 and Table 1 ).
Prior to transfection cells should be maintained in an appropriate
growth medium at logarithmic phase of growth. Nucleic acids may
be introduced into eukaryotic cells using various chemical, lipid or
physical methods ( 1 5 ) .The ef ciency of each method will vary
depending on the cell line and vector used, and the transfection
3.2. Transfection,
Cloning, and Selection
of Eukaryotic Cell
Lines
3.2.1. Transfection
142 B. Wognum and T. Lee
conditions will need to be optimized for each cell line. After
transfection, cells should be incubated in growth medium without
antibiotics for 2448 h before commencing the selection and
cloning procedure.
Transfection ef ciency and cell survival depend on a number of
factors including the gene transfected, the cells used and the trans-
fection method employed. Optimal cell numbers per plate need to
3.2.2. Cloning and
Selection in ClonaCell-TCS
or ClonaCell-CHO
Fig. 3. Procedure for transfected cell line selection and cloning in semisolid medium.

143 9 Cloning in Semi-solid Media
Table 1
Suggested antibiotic concentrations required for selection of transfected cell lines
in ClonaCell-TCS medium
Cell line
Antibiotic BAF3 Molt4 K562 Jurkat Daudi UT-7 FD5 CHO-S
Puromycin ( g/mL) 1 0.5 N.D. 0.2 0.25 0.3 N.D. N.D.
G418 (mg/mL) 1 2 1.2 1 1 0.5 0.5 1.6
Hygromycin (mg/mL) N.D. 0.5 1 0.5 0.15 0.3 N.D. N.D.
be determined for different cell lines and applications. An example
of the conditions and concentrations required for 1 10
7
trans-
fected cells (ten 10 cm plates at 10
6
cells/plate) is described
below.
1. Thaw ClonaCell-TCS or ClonaCell-CHO medium overnight
at 28C. Warm the bottle to RT before proceeding to next
step.
2. Shake bottle of ClonaCell medium vigorously to mix contents.
Let bubbles rise the top (approximately 10 min).
3. The antibiotic used for selection of stable transfectants will
vary depending on the selectable marker present on the plasmid
used in the transfection. The concentration of the selective
agent required will need to be determined for each cell line and
will need to be performed in the medium used for the selection
of transfectants. Prepare antibiotic solution: Add 10 nal
antibiotic concentration required for selection (see Note 10).
Any other compounds, e.g., cytokines (if required) can be
added to the growth medium, but the total volume should not
exceed 10 mL.
4. Using a 12-mL syringe and 16-gauge blunt-end needle, dis-
pense 8 mL of ClonaCell medium to each of ten 14 mL Falcon
sterile tubes. Add 1 mL of the 10 antibiotic and any other
supplements to the 8 mL of ClonaCell-TCS in each tube. Mix
tubes thoroughly by inverting several times (see Note 7).
5. Harvest the transfected cells into a 50 mL conical tube and
centrifuge for 10 min at 400 g . Resuspend the cells in a total
volume of 10 mL liquid growth medium without antibiotic.
6. Plating at several cell densities is recommended as transfection
ef ciency may vary from experiment to experiment. Optionally,
to plate cells at different plating densities (e.g., 2 10
6
, 1 10
6
,
and 0.5 10
6
cells per plate), resuspend the cells in a total
144 B. Wognum and T. Lee
volume of 5 mL, remove 2 mL, and add 1 mL to each of two
tubes with medium suspension (see step 7 below). To the
remaining 3 mL of cell suspension add 3 mL of liquid growth
medium, mix well, and remove 3 mL, and add the cell suspen-
sion to each of three tubes of semisolid medium (step 7).
To the remaining cell suspension add 23 mL of liquid growth
medium, mix well, and dispense 1 mL to each of the remaining
ve tubes with culture medium (step 7).
7. Add 1 mL of cell suspension to each tube. Mix tubes thor-
oughly by inverting several times and let sit for 15 min to allow
air to rise to the top. Thorough mixing is essential to ensure
thorough distribution of antibiotics and cells throughout the
viscous methylcellulose-based cloning medium.
8. Using 12-mL syringes and 16-gauge blunt-end needles, asep-
tically plate out 9.5 mL from each tube into separate 10 cm
sterile petri dishes. Tilt the plates gently to level mixture, being
careful to avoid trapping of air (see Note 7).
9. Incubate plates at 37C in 5% CO
2
and >95% humidity. Do not
disturb plates for 714 days. The time required to see macro-
scopic colonies varies depending on the cell line used for trans-
fection and the antibiotic used for selection.
1. After 714 days of culture, depending on the cell type and
antibiotic used for selection, examine the plates for the pres-
ence of colonies that are visible to the naked eye. Place plates
in a biosafety cabinet and aseptically remove isolated colonies
from the plates using a pipettor set to 10 L, and sterile pipette
tips. Pipette each clone into an individual well of a 96-well
tissue culture plate containing 200 L of growth medium
containing the speci c antibiotic used in the selection process.
Incubate the plates at 37C in 5% CO
2
for 14 days without
feeding. Alternatively, an automated colony harvester may be
used (see Note 8). It is recommended to pick colonies of
different sizes, as slower growing transfectants which produce
smaller colonies may be very good protein producers. Such
slow growing transfectants are often missed in other transfec-
tion screening procedures. Usually by the fourth day, each well
has a high cell density and the medium has begun to turn
yellow.
2. Transfer 150 L of each cell suspension to a separate well on a
96-well plate and assay for expression of the desired transfected
gene product (e.g., ELISA, Flow Cytometry, Western Blotting,
etc.). Add 150 L of fresh growth medium containing anti-
biotic to every well of the original plate.
3. Transfer 2 100 L of cell suspension of the positive clones
identi ed in step 2 to each of two wells of a 24-well plate
containing 1 mL of growth medium and antibiotics.
3.2.3. Harvest
and Screening
145 9 Cloning in Semi-solid Media
4. When cells have grown to a suitable density, cryopreserve
the cells from one well and expand the other in increasing
volumes of growth medium. If desired, the concentration of
antibiotics in the culture media may be decreased or even
eliminated after several passages. However, some transfectants
are unstable and need selective pressure to be maintained, or
need to be recloned periodically. The cryopreserved cells serve
as backup in case the cultured cells are lost, or in case protein
expression is lost and subcloning of the cultured cells is
unsuccessful.

1. Selection and cloning of transfected cell lines in semisolid
medium works best with cell lines that can grow as nonadherent
cells in suspension cultures or that can be adapted to growth
under nonadherent conditions. The following nonadherent
cell lines have been found to grow well in semisolid ClonaCell-
TCS medium: BaF/3 (murine pro-B cell), Molt-4 (human T
cell lymphoma), K562 (human myeloid-erythroid cell), Jurkat
(human T lymphoblastoid), Daudi (human B lymphoblastoid),
UT-7 (human T cells), and FD-5 (murine pre-myeloid).
In addition various adherent cell lines have been shown to
adapt well to nonadherent culture conditions and to form
colonies in semisolid medium (i.e., BHK-21, CHO, HEK293).
Although nonadherent cell lines are more likely to grow in
semisolid media, not every nonadherent cell line grows as well.
For example, the TF1, KG1, and M1 cell lines dont form
distinct colonies after plating in ClonaCell-TCS medium.
If there is no prior knowledge of the ability of a cell line to
grow in semisolid medium or the cell line will be used with a
different semisolid medium than used in earlier experiments, it
is recommended to test the ability of the cell line to grow as
nonadherent colonies in the semisolid medium before using
this cell line in transfection experiments (see Note 11). Non-
tissue culture treated plastic ware should be used.
2. Adaptation of cell lines to growth in the same base medium as
that used in the semisolid medium may be necessary for opti-
mal plating ef ciency. Culture media from different suppliers
have different compositions and ability to support clonal
growth of different cell lines. Cells that have been cultured in
a speci c medium will over time result in a unique subpopula-
tion with optimal growth in that particular medium. These
cells may adjust poorly to a rapid switch to a semisolid medium
with different media composition, but may grow better if
4. Notes
146 B. Wognum and T. Lee
adapted gradually to the same liquid medium as used in the
semisolid medium. Adaptation prior to plating in semisolid
media is particularly important when serum-free or protein-
free media are used, as cells grown in such media often have
slower growth rate and lower cloning ef ciency than cells
grown in serum-containing media. Serum-free and protein-
free media (e.g., ClonaCell-CHO-ACF and ClonaCell-
CHO-CD) are often preferred for cell line development, in
particular for expression of recombinant proteins with poten-
tial therapeutic applications. Cells can be adapted slowly to a
new medium by gradually increasing the relative volume of
new medium during consecutive passages. The growth rate
and cell viability should be closely monitored at each passage.
Some reduction in growth rate may be expected when switch-
ing cells from a serum-containing medium to a serum-free or
protein-free medium. The cloning ef ciency may also decrease
as the growth of cell lines in serum-free and protein-free media
may be more dependent on cell density than in serum-containing
media. After transfection the cells may need to be plated at
higher cell densities than cell lines grown in serum-containing
media to ensure adequate colony numbers. However, the
number of colonies is also determined by the ef ciency of
the transfection and antibiotics selection. As these variables are
often unknown it is recommended to plate newly transfected
cells at a range of cell densities.
3. The procedures and ClonaCell-HY media for selection and
cloning of hybridoma cell lines described in this chapter have
been optimized for mouse hybridomas. Selection and cloning
in semisolid media may also be useful for generation of mono-
clonal antibodies from other species, speci cally rats, rabbits,
and hamsters, but different medium formulations and/or
culture conditions may need to be selected. If established
hybridomas from these species are already available the useful-
ness of ClonaCell-HY for selection and cloning hybridomas
from these species could be tested in a cloning experiment
(see Note 11). The formulation that gives highest recloning
ef ciency (preferably >50%, but lower ef ciencies may be
acceptable), and that supports the development of distinct and
large colonies with good morphology, consistency (i.e., not
runny or hazy) and high cell viability after plucking can then be
used for selection and cloning of newly generated hybridoma
cell lines.
4. If a suitable liquid culture medium formulation is already used
in ones lab for selection and cloning of transfected cell lines in
suspension cultures one could prepare a semisolid version of
this medium by combining the liquid medium with a stock
solution of culture-tested methylcellulose, (e.g., MethoCult
147 9 Cloning in Semi-solid Media
H4100 or M3134, STI), It may be useful to prepare several
media, each containing a different methylcellulose concentra-
tion (e.g., between 0.8 and 1.2%), and test these media in a
cloning experiment using nontransfected cells (see Note 11).
The medium formulation that gives highest cloning ef ciency,
best colony size and morphology, and highest cell viability can
then be used for selection and cloning of new transfectants.
5. It is important to remove all the serum adhering to the cells,
by washing with serum-free Medium B. If the serum is not
removed, the PEG will not fuse the cell membranes and the
fusion frequency will drop drastically.
6. It is important to completely break up the cell pellet prior to
adding PEG in order to ensure ef cient fusion of the cells.
During this procedure, not all cells will form a pellet, as some
will clump in the PEG. Do not aspirate the clumped cells. Work
quickly since cells must not be exposed to PEG for too long or
cell viability will drop.
7. Methylcellulose is a viscous solution and cannot be accurately
dispensed using pipettes due to adherence of the medium to
pipette walls. Syringes with blunt-end needles, rather than reg-
ular hypodermic needles should be used for aliquoting the
media to prevent needle-stick injuries. It is recommended to
put the plates in a separate plastic container together with an
open 100 mm petri dish containing 10 mL sterile distilled
water to maintain moisture content in methylcellulose cultures.
Open and close the incubator door carefully to avoid shaking.
It is important not to disturb the plates for the rst 10 days as
this may result in dispersed colonies.
8. Manual picking of colonies from the semisolid medium is the
most time- and labor intensive step of the cloning and selec-
tion procedure. In a typical experiment several hundreds of
colonies can be obtained and it may not be possible to harvest
each colony present in each culture dish. An automated colony
harvester, such as the ClonaCell EasyPick (catalog # 30000,
STI) may be used to reduce the amount of manual manipula-
tion of the cultures and increase the number of colonies that
are harvested. Alternatively, the procedures as described in
Subheadings 3.1.4 and 3.2.2 can be modi ed to enable
screening of clones prior to plucking and thus reduce the total
number of colonies that need to be plucked. In this modi ed
procedure the cells are not plated in 10 cm dishes after
resuspending in the semisolid medium as described in
Subheading 3.1.4 , step 3 and Subheading 3.2.2 , step 7, but
instead are distributed over individual wells of 96-well plates at
6080 L per well. After culture, the wells that have colonies
in them, or alternatively all wells, are carefully overlaid with
150 L of liquid medium (Medium E for hybridomas; an
148 B. Wognum and T. Lee
appropriate liquid medium for transfected cell lines). The cultures
are incubated at 37C for 2 days to allow antibodies or recom-
binant proteins to diffuse into the liquid medium. A fraction of
the overlaid liquid medium (e.g., 100 L) is carefully removed
without disturbing the colonies in the semisolid medium and
tested in appropriate screening assays. Colonies in positive
wells are then harvested and expanded following the standard
procedures. A major advantage of this procedure is that only
colonies in positive wells need to be harvested. A disadvantage
is that there may be more than one clone in a single well. If this
is the case, each colony needs to be harvested from the positive
wells, expanded and rescreened to identify which clone is
positive. If the colonies overlap and it is not possible to harvest
them separately from the well, it will be necessary to reclone
the cells to ensure that a monoclonal cell line is obtained that
produces the antibody or recombinant protein of interest.
9. Newly generated hybridomas and transfected cell lines may be
recloned to select for subclones that have better growth rate
and/or higher protein production than the parental clone.
The plating ef ciency of different hybridomas and transfected
cell lines may be variable. Therefore, a range of plating densi-
ties should be used, (e.g., 100, 500, 1,000 cells per 10 cm
dish) to ensure that at least one of the dishes will yield enough
individual colonies for harvesting and testing and will not be
overplated.
10. Before selection and cloning of transfected cell lines in semi-
solid media it is important to determine the minimum anti-
biotic concentration that is lethal for non-transfected cells.
The optimal antibiotic concentration will have to be deter-
mined by titration of the antibiotic in a cloning experiment
using non-transfected cells (see Note 9). It is not suf cient to
use information from liquid cultures as antibiotic sensitivity
may be different between liquid and semisolid culture condi-
tions and may also be dependent on the medium formulation.
Nontransfected cell should be plated at approximately the same
density that will be used when selecting for transfectants, or
the antibiotics titration could be done at a range of plating
densities to ensure that useful results will be obtained. Each set
of cultures should be supplemented with a range of different
concentrations of the appropriate antibiotic (e.g., G418 or
hygromycin, dependent on the selectable marker in the vector
chosen for transfection). After culture, the number of colonies
that are visible to the naked eye should be counted and the
minimal antibiotic concentration required to kill all the cells
established. This is the antibiotic concentration that should be
used to select the cells after transfection. An example of typical
antibiotic concentrations used for selection of different cell
149 9 Cloning in Semi-solid Media
lines in ClonaCell-TCS is shown in Table 1 .These data are only
meant as indication of the concentration ranges for different
antibiotics. The growth characteristics of cell lines may change
over time, and antibiotics from different sources may have
different activities. Therefore the minimum lethal antibiotic
concentration should be determined in each lab using the cell
line, culture media, and antibiotic preparations selected for use
in the subsequent transfection experiments.
11. To test the growth of a cell line in semisolid medium, non-
transfected cells are plated at a range of cell concentrations,
e.g., 100, 500, and 1,000 cells per 10 cm petri dish. After
culture colonies are counted and the plating ef ciency is
calculated according to the following formula: Plating
ef ciency = number of colonies counted 100%/number of
cells plated. If the plating ef ciency is high at a range of plating
densities, e.g., >20%, and the colonies are distinct and non-
overlapping, it is likely that the cell line will form colonies after
transfection, assuming that other conditions (speci cally, trans-
fection ef ciency, antibiotic concentration) are adequate as
well. If a cell line produces no or only very few colonies in the
semisolid medium, even at high plating densities, it may require
a semisolid medium with different composition that better
supports clonal growth of these cells or it may need to be
adapted rst to growth in a liquid version of the semisolid
medium to be used in transfection and cloning experiments
(see Notes 24).
References
1. Sambrook J, Fritsch EF, Maniatis T (1989)
Molecular cloning: a laboratory manual, 2nd
edn. Cold Spring Harbor Laboratory Press,
New York
2. Carey M, Smale ST (2000) Transcription reg-
ulation in eukaryotes: concepts, strategies, and
techniques. Cold Spring Harbor Laboratory
Press, New York
3. Kreigler M (1990) Gene transfer and expression:
a laboratory manual. Stockton Press, New York
4. Ravid K, Freshney RI (1998) DNA transfer to
cultured cells. Wiley, New York
5. Dasso M (2005) Expression and introduction of
macromolecules into cells. In: Bonifacino JS,
Dasso M, Harford JB (eds) Current protocols in
cell biology. Wiley, New York, pp 20.0.120.0.2