Clonal Cultures in vitro for
Haemopoietic Cells Using
Semisolid Agar Medium
An exponential increase in knowledge of the regulation
of haemopoiesis has been witnessed since
the mid-1980s. Over 20 cytokines (colony-stimulating
factors, erythropoietin, thrombopoietin, and interleukins)
have now been identified, molecularly cloned,
and expressed. Many of these recombinant haemopoietic
growth factors are now being used in clinical situations
where they have been found to be useful in
correcting anaemia and white cell deficiencies either in
chronic-inherited disease or following acute treatment
(e.g., chemotherapy, bone marrow transplantation)
(Atkinson, 1993; Clark and Kamen, 1987; Mertelsmann
et al., 1990; Sheridan et al
The discovery of haemopoietic growth factors was
facilitated greatly by the ability to grow haemopoietic
cells in vitro
. These culture systems enable the undifferentiated
haemopoietic precursors to proliferate and
differentiate into various haemopoietic cell lineages.
Especially valuable has been the development of
clonal cultures using semisolid agar or methylcellulose
culture medium for haemopoietic precursor cells. In
the presence of appropriate growth factors, these precursor
cells proliferate and produce a clonal colony of
differentiated cells. This will allow biological, viral,
biochemical, or molecular studies to be performed
on individual cell clones. In addition, by counting
colonies, it is also possible to infer the number of precursor
cells in the starting cell population. This is possible
because a linear relationship exists between the
number of colonies formed and the number of cells
cultured. By comparison, liquid suspension cultures of primary haemopoietic cells do not allow enumeration
of precursor cell numbers as the progeny are intermingled
in the culture dish. The second feature of
clonal cultures is the dose-response relationship that
exists between the amount of growth factor and the
number of colonies stimulated. This dose-response
relationship is sigmoid, having a linear phase and a
plateau phase. The linear portion of the curve can be
used to determine the amount of growth factor activity;
in cultures described here, 50 units of growth factor
activity correspond to the amount of activity stimulating
50% of maximal colony numbers. More detailed
information on haemopoietic colony formation and
cytokines can be found in Metcalf (1984, 1985, 1986,
1991) and Nicola (1991).
II. MATERIALS AND
A. Semisolid Agar Medium Cultures
Iscove's modified Dulbecco's medium powder, with
4mM L-glutamine and 25 mM HEPES buffer (IMDM,
Cat. No. 12200-085 for a 5-1itre batch), is from GIBCOInvitrogen.
DEAE-dextran hydrochloride (Cat. No.
D9885), L-asparagine monohydrate (Cat. No. A4284),
2-mercaptoethanol (Cat. No. M7522), penicillin G (Cat.
No. P7794), and streptomycin (Cat. No. $9137) are
from Sigma-Aldrich. Bacto agar is from Bacto Laboratories
(Cat. No. 0140-17). Recombinant haemopoietic
growth factors and cytokines can be purchased from a number of commercial suppliers (e.g., PeproTech EC
Ltd, R&D Systems). For routine cultures, conditioned
medium can be prepared from a number of tissues (see
later). Foetal calf serum (FCS) can be obtained from a
number of suppliers, but requires pretesting (see later).
Bacteriological graded petri dishes (35-36 and 100mm
diameter) can be obtained from a number of suppliers
as a tissue culture grade dish is not required. For viable
cell count, eosin Y from Sigma-Aldrich (Cat. No.
E4009) is required. Distilled Milli-Q endotoxin-free
0) is routinely used.
B. Conditioned Medium
IMDM (Cat. No. 12200-085) and pokeweed mitogen
(PWM, Cat. No. 15360-019) are from GIBCOInvitrogen.
2-Mercaptoethanol (2ME, Cat. No. M7522),
sodium bicarbonate (NaHCO3
, Cat. No. $6014), penicillin
G (Cat. No. P7794), and streptomycin (Cat. No.
$9137) are from Sigma-Aldrich. FCS can be obtained
from various suppliers, and batches can only be
selected by prior testing. If a batch is available that is
known to support good colony formation, then this
can be used to prepare conditioned media. A variety
of flasks can be used to prepare PWM-stimulated
spleen cell-conditioned media (PWM-SCM) for murine
cultures, although we routinely use 1- to 2-1itre glass
flasks fitted with a cotton plug to allow gas diffusion.
For preparation of human placental conditioned
media, a variety of flasks can also be used, but we routinely
use disposable 75-cm2
tissue culture flasks (Cat.
No. 25110-75, Corning).
C. In Situ Colony Staining
Glutaraldehyde solution (grade II, 25% in water,
Cat. No. G6257), ethanol (Cat. No. 02862), urea (Cat.
No. U6504), acetylthiocholine iodide (Cat. No. A5751),
sodium phosphate dibasic (Na2
, Cat. No. 71629),
sodium phosphate monobasic (NaH2
, Cat. No.
71492), sodium citrate (Cat. No. $1804), copper(II)
, Cat. No. C1297), potassium
, Cat. No. 60310], and Mayer's
haematoxylin staining solution (Cat. No. MHS-16)
are from Sigma-Aldrich. Luxol fast blue MBS (Code
Pack No. 10732) is from NBS Biologicals. Hardened
ashless 541 filter paper of 5.5-cm diameter (Cat.
No. 1541 055) is from Whatman, and DePex (D.P.X)
neutral mounting medium (Cat. No. 3197) is from
Bacto Laboratories. Plain 2 × 2-in. microscope
slides (Cat. No. 5075) and 45 × 50-mm cover glasses
(Cat. No. 4550-1) can be obtained from Brain Research
For colony examination and counting, a zoom stereomicroscope
(Model SZ4045) fitted with a clear stage
plate and a base illuminator from Olympus is used
routinely. Many brands of CO2
incubators are available
and it is recommended that one with stainless-steel
water jackets be obtained. In addition, to minimize the
desiccation of cultures, an incubator without an inbuilt
fan is preferred. A standard light microscope equipped
with 10 and 40× objectives will be sufficient for routine
cell counting and in situ colony typing. Graduated,
glass blow-out pipettes in volumes of 1, 5, 10, and
25 ml are routinely used, although they can be replaced
by disposable pipettes. For the concentration of conditioned
media, we use an Amicon hollow-fibre concentrator
(Model DC2A) fitted with a HIP10 membrane.
A. Method for Establishing and Scoring
The fundamental steps in establishing agar-medium
cultures are fourfold: (1) mix equal volumes of doublestrength
medium and double-strength agar solution,
(2) add the cells to be cultured and mix, (3) pipette the
cell suspension onto the culture dishes, and (4) after
gelling the culture dishes are put in an incubator for
- IMDM for agar cultures (AIMDM): 88.3 g IMDM,
0.6g penicillin G, 0.375g streptomycin, 7.5 ml DEAEdextran
(50mg/ml solution), 1.0g L-asparagine, 24.5g
NaHCO3, and 29.5 µl 2-mercaptoethanol. Dissolve the
contents of a 5-1itre package IMDM (88.3 g) in I litre of
H2O using a magnetic stirrer for mixing. Rinse the
inside of the package to remove all traces of powder.
Add afore-listed reagents while stirring continuously.
Add H2O to a final volume of 1.95 litres and gas
medium with 100% CO2 until it is a yellow-orange
colour. The prepared endotoxin-free medium should
then be filter sterilized and distributed in 100- or 250-
ml aliquots, which are then tightly capped, stored at
4°C and protected from light. Each preparation of
AIMDM should be batch tested against one that is
currently in use if applicable.
- Agar: 0.6 g Bacto-agar and 100ml H2O. Weigh
agar into a 100-ml flask, add 100ml H2O, and plug
flask loosely. Bring to boiling for 2min over a gas
flame. Prepare immediately before setting up cultures and maintain at 45°C in a water bath. Each new lot of
Bacto-agar should be batch tested against one that is
currently in use if applicable.
- Foetal calf serum: FCS is used as a source of nutrients
in cell cultures. Batches of FCS should be tested
extensively prior to purchase, for optimal colony formation
in colony number and colony size, in semisolid
cultures. They should also be titrated to determine the
optimal concentration (final concentration is usually
5-20%). The storage/shelf life of FCS at -20°C is at
least 2 years and at-70°C it as long as 10 years. Centrifugation
may be necessary to remove any sediment
that forms after thawing. It is otherwise ready for use.
Heat inactivation is optional but not necessary.
- Eosin for viable cell counts: Prepare stock solution
[10% eosin-yellow powder (w/v)] in normal saline
and keep at 4°C. Mix 0.2 ml of stock eosin solution with
8.6 ml normal saline and 1.5 ml FCS to prepare working
solution. Aliquots of the working solution should be
- Hemopoietic growth factors: If purchased commercially,
they should be pretested to determine the
amount required for optimal colony formation. If
conditioned media are prepared (see later), they also
require titration to determine the optimal concentration
for maximal colony formation without evidence
of high-dose inhibition. Stimuli should be divided into
aliquots at a concentration at least 10-fold higher than
that to be used finally in the culture dish. They can be
stored frozen, but once thawed, they should not be
frozen again as this can result in a loss of activity.
- Single-strength Iscove's modified Dulbecco's medium:
Dissolve the entire contents of a 5-1itre package IMDM
(88.3g) with 4.8 litres of H2O and mix with gentle
stirring. Rinse the inside of the package to remove all
traces of powder. Add 15.12g NaHCO3, 29.5H µl 2-
mercaptoethanol, 150 mg penicillin G, and 100 mg
streptomycin while stirring continuously. Add H2O to a
final volume of 5 litres and gas medium with 100% CO2 until it is a yellow-orange colour. The prepared endotoxin-
free medium should then be filter sterilized and
distributed in 100- or 500-ml aliquots, which are then
capped tightly, stored at 4°C and protected from light.
- Pokeweed mitogen: This should be prepared immediately
prior to use. Any material not used should be
discarded. Make up powder with 5ml of doubledistilled,
deionized water. Remove from the vial and
dilute 1 : 15 (v/v) with H2O.
- Warm AIMDM to room temperature.
- Prepare agar solution.
- Count viable cells using a haemocytometer and
eosin as dye.
- Draw culture layout, in a book, showing culture
number, stimuli for each culture dish, and the number
of cells for each culture dish.
- Place required number of culture dishes on incubator
trays and number lids individually according to
the culture book.
- Add required stimuli to appropriate culture
dishes as described in the culture book. For each
culture, the required amount of stimulus is usually
added in 0.1 ml per culture dish. This amount can be
less but should not exceed 0.2ml as the agar may not
- For agar cultures with 20% FCS final concentration,
mix AIMDM (three parts) and foetal calf serum
(two parts) first and then add an equal volume of agar
(five parts). Cells are added last to this single-strength
agar medium, which should be now roughly at
36-37°C before plating. For each group of cultures,
allow 1 ml of agar medium each per culture plus at
least 1 ml extra for wastage in pipetting. If by pretesting,
a lower concentration of FCS is sufficient for
optimal colony formation, replace the leftover volume
with H2O so that the amount of AIMDM and FCS
equals 0.5 ml for each 1 ml of culture.
- Aliquot 1-ml volumes into petri dishes and swirl
to mix stimuli and agar medium-containing cells.
- Allow mixture to gel and place in a fully humidified
containing 5-10% CO2 in air.
- After the required incubation period (normally 7
days for murine progenitor cells and 14 days for human
progenitors and murine high-proliferative stem/progenitor
cells), remove cultures from incubator and
count colonies using an Olympus SZ stereomicroscope
at 30-35× magnification. Murine haematopoietic
colonies are defined routinely as clones greater than
50 cells and human colonies greater than 40 cells
(some investigators count human colonies as having
greater than 20 cells). Place the microscope on top of
a black platform and adjust the concave side of the
mirror until cells appear white against a black background
(Fig. 1). To aid in the enumeration of colonies,
we routinely put the 35-mm agar culture dishes on top
of an inverted 60-mm culture dish marked with 6-mm2 grids.
|FIGURE 1 Photomicrograph of a dispersed
B. Preparation of Conditioned Media
Although specific stimuli may be required for many
situations, and always for use as a positive control, a
variety of conditioned media containing mixtures
of haemopoietic growth factors can be prepared. The
following examples provide descriptions for the
preparation of two conditioned media: one suitable for
human cultures and the other for murine cultures.
1. Preparation of Human Placenta-Conditioned
2. Preparation of Murine Pokeweed Mitogen-
Stimulated Spleen Cell-Conditioned Medium
- Placenta should be obtained within 9 h of birth.
Place it on a large sterile tray in a biological safety
- Using sterile instruments, remove outer layer of
placenta. Assume that this portion is not sterile. Instruments
can be kept sterile by periodically returning
them to boiling water.
- Cut a portion (1 cm3) of exposed placenta and
place in a 100-mm petri dish containing 10ml IMDM.
Limit each petri dish to 8-10 pieces.
- Having removed sufficient pieces of placenta,
rinse each piece through three changes of IMDM in
100-mm petri dishes to remove most of the blood.
- Place 18-20 pieces of placenta into tissue culture
flasks (75cm2, Cat. No. 25110-75, Coming) in 60ml
IMDM with 5% (v/v) FCS.
- Place the placenta cultures, with the caps sealed
loosely, in a 37°C fully humidified incubator containing
5-10% CO2 in air.
- After 5-7 days of incubation, harvest the
medium free of placenta by pouring the contents
of each flask through cotton gauze into a collection
flask. Centrifuge the medium at 3000g and store
the supernatant at -20°C until 4-5 litres has
- Concentrate the placenta-conditioned medium
approximately 10-fold using a hollow fibre concentrator
(this type of concentrator is preferred because of
the relatively large volumes involved).
- Filter sterilize the concentrate and test by titration
using human bone marrow semisolid agar cultures.
Select the batches of conditioned media that
display a sigmoid dose-response relationship with the
number of colonies formed and without evidence of a
III. COLONY TYPING
- Prepare a single-cell suspension of murine spleen
cells, either by teasing the spleen tissue with needle or
by forcing it through a fine stainless steel mesh.
- Place the spleen cells in a tube and allow them to
stand for 5rain to allow larger tissue fragments to
sediment. Remove the supernatant and determine
viable cell numbers.
- Make up the cells to 2 × 106/ml in IMDM containing
10% FCS. (The concentration of FCS should be
as low as possible and can be determined only by preliminary
testing.) Add pokeweed mitogen (0.05 ml of a
1:15 dilution of freshly prepared stock is added for
each millilitre of culture medium).
- Incubate the cells in medium for 7 days at 37°C in a fully humidified incubator containing 5-10% CO2 in air. The cells can be incubated in a variety of
containers. We routinely use 2-1itre flasks, with cotton
plugs, containing 250ml of medium.
- After incubation, harvest the conditioned
medium and centrifuge at 3000g to remove cellular
debris. Concentrate the medium 10-fold as described
earlier for human placenta-conditioned medium.
- Titrate the concentrated PWM-SCM using cultures
of routine bone marrow cells to determine the
concentration required to give plateau numbers of
- The conditioned medium can then be diluted to
a concentration 10 times that is required for maximal
colony formation, divided into 20-ml aliquots, and
stored at-20°C until required. Once thawed the PWMSCM
should be stored at 4°C.
Colonies grown in agar cultures can be typed using
an in situ
whole plate staining sequentially with Luxol
Fast Blue to detect eosinophil granules (Johnson &
Metcalf, 1980), acetylcholinesterase to detect megakaryocytes
(von Melchner and Lieschke, 1981) and
Mayer's haematoxylin for nuclear morphology to
detect neutrophils and monocyte/macrophages.
Preparation of Fixative and Staining Solutions
A 2.5% (v/v) glutaraldehyde in PBS is prepared by
mixing 1 part of 25% glutaraldehyde with 9 parts of
PBS. The luxol fast blue staining solution is prepared
by dissolving 0.1 g powdered luxol fast blue MBS dye
(NBS Biologicals Ltd., Hungtingdon, Cambs, England)
in 100ml of 70% ethanol saturated with urea. The
substrate solution for acetylcholinesterase staining
should be prepared fresh each time by dissolving
10mg acetylthiocholine iodide (Sigma-Aldrich) in
15 ml of 100 mM
sodium phosphate buffer, pH 6.0. One
millilitre of 100mM sodium citrate, 2ml of 30mM
copper sulphate, and 2ml of potassium ferricyanide
solutions are then added sequentially with constant
- Add 2ml 2.5% glutaraldehyde into individual
culture dishes and leave at room temperature
- Transfer the gel onto a 3 × 2-in. glass slide with
the aid of a water bath and then cover the gel with a
wet 5.5-cm Whatman 541 filter paper and allow drying
at room temperature in a fume hood.
- Remove the filter paper, leaving a thin film of gel
containing compressed colonies on the glass slides.
- Slides are first stained for acetylcholinesterase by
incubating the slides in the dark with the substrate
solution at room temperature for 3 h.
- After washing under running tap water for 10-
15 min, transfer slides into the luxol fast blue staining
solution and stain for 30min at room temperature.
- After another washing under running tap water
for 30min, counterstain slides with Mayer's haematoxylin
for 1 min, wash, and "blue" in running tap
- Dry slides at room temperature, mount with
DePex, and examine under a light microscope after
- Numerous pitfalls are associated with these procedures.
A major problem involves selection of a suitable
batch of FCS. If possible, a known positive sample
should be obtained from a colleague or a commercial
source (e.g., StemCell Technologies) for use as a control
when testing new batches.
- The agar needs to be boiled to ensure that it is
dissolved properly. For this reason, it is recommended
to use a gas flame. The agar will initially bubble up when boiliing, and care must be taken to prevent it
from overflowing. Once the bubbling has subsided, the
agar should be boiled for another minute. An autoclave
should not be used to prepare the agar solution.
- It is imperative that the incubator being used is
fully humidified, as desiccation of the cultures will
prevent colony growth. With satisfactory cultures, a
small volume of liquid will be evident at the edge of
the agar medium when the cultures are tilted. If desiccation
has occurred, the surface of the agar medium
will display irregularities instead of being smooth and
shiny. To prevent desiccation, the incubator should
contain one or more large open trays containing H2O.
Humidity can be improved by pumping the air-gas
mixture into the incubator via a tube immersed in one
of the trays of water. Many incubators are fitted with
a fan to produce a uniform atmosphere with the closed
incubator. This can also cause the desiccation of cultures
and the fan may have to be disconnected.
- The possibility of cultures drying out is increased
by extending the incubation time. For cultures in
excess of 7 days it is a good policy to place culture
dishes in a 100-cm petri dish (two cultures per dish)
containing a third open-lid, 35-mm culture dish with
H2O. It is also a good policy to minimize the number
of openings of the incubator.
- When establishing the cultures, problems can
arise due to the temperature of the agar-medium
mixture. If too cold, it will gel prematurely and the
cells will not be immobilized properly. If too hot, it
will kill the cells. It is recommended that the agar be
maintained at 45°C and that the AIMDM and FCS be
allowed to warm to room temperature (18-20°C). When agar, medium, and FCS are mixed, the temperature
of the solution will be about 37°C which will not
kill the cells but will still be above the gelling temperature
of the agar. Once cells have been added to the
agar medium mixture and mixed, it should be dispensed
to culture dishes as soon as possible. As 0.1 ml
of stimulus is usually present in the culture dish, the
mixture should be swirled gently to allow homogeneous
mixing. All these actions should be executed
before gelling starts to occur.
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