Three-Dimensional Cultures of Normal
and Malignant Human Breast Epithelial
Cells to Achieve in vivo-like
Architecture and Function
Apicobasal polarity, properly positioned cell-cell
contacts, and attachment to basement membrane are
fundamental characteristics of simple glandular
epithelia, such as the mammary gland. The development
and maintenance of this polarized structure are
essential for the formation of tissue architecture,
control of proliferation, and the differentiated function
of epithelial cells (Roskelley et al.
, 1995). Loss of architectural
intactness and polarity is one of the pathological
hallmarks of epithelial carcinoma (Bissell and
Radisky, 2001). Although traditional studies of epithelial
cells grown as a monolayer on tissue culture plastic
remain a powerful tool to dissect and understand the
molecular events of signaling machineries, tissue
culture plastic does not recapitulate the microenvironment
or morphology of glandular epithelium in vivo
Animal models have provided us with invaluable
insights, which have led to a greater understanding
of the events involved in mammary morphogenesis
and tumorigensis in vivo
, and aid in the translation
from basic cellular research into clinical application.
However, the complexity of animal model systems
prevents us from precisely pinning down the specific
biochemical and cell biological pathways involved
in mammary morphogenesis and tumor formation.
Therefore an in vitro
cell-based model system that provides
epithelial cells with an in vivo
recapitulates both the three-dimensional (3D) organization and multicellular complexity, and is
conducive to systematic experimental pertubation is
optimal to bridge the gaps between epithelial monolayer
cultures and animal models.
We have developed an assay in which primary or
phenotypically normal human breast epithelial cells
cultured in a laminin-rich basement membrane (lrBM)
undergo a three-dimensional reorganization to form
structures that mimic in vivo
acinar structures in
culture (Petersen et al.
, 1992). LrBM, available commercially
as Matrigel, is a mixture of basement membrane
proteins that include ~80% laminin, ~10% type
IV collagen, entactin, and ~10% heparin sulfate proteoglycan,
derived from Engelbreth-Holm-Swarm
mouse tumor (Orkin et al.
, 1977; Kleinman et al.
1987). The malleability of this specialized gel and its
ability to signal through integrins, as well as other
ECM receptors, induce changes in the cells, which
allow them to withdraw from the cell cycle, organize,
and form a central lumen. The entire acinar structures
achieve apical basal polarity, and cells within the acini
express tissue-specific genes, processes that are not
easily accomplished by the same cells cultured on conventional
tissue-culture plastic. We refer to the assay
as 3D BM assay. The following are advantages of 3D
BM assays in studying normal and malignant cells. (1)
Because the cells are able to form tissue-like structures,
mechanisms of tissue specificity can be studied ex vivo
These cultures are amenable to a variety of perturbations
and manipulations that can be used to understand
how cells may signal in vivo
. (2) The assay allows breast tumor cells to be distinguished easily from their
nonmalignant counterparts because the tumor cells
fail to stop proliferating and do not become organized
into tissue-like structures (Petersen et al.
, 1992; Weaver et al.
, 1996). (3) Addition of specific signaling inhibitors
or blocking antibodies or delivery of genes to the
tumorgenic cells can be used to analyze signaling pathways
involved in the acquisition of the nontumorgenic
phenotype (Weaver et al.
, 1997; Wang et al.
, 1998, 2002).
(4) The 3D BM cultures can be used to understand
the novel roles of oncogenes and tumor supressors
genes (Howlett et al.
, 1994; Spancake et al.
Muthuswamy et al.
, 2001; Debnath et al.
, 2002). (5)
These assays can assess the response of tumorigenic vs
nontumorigenic cells to potential therapeutics and
unravel novel mechanisms (Weaver et al.
, 2002). (6) The
assays (3-10 days) can be performed rapidly and in a
high throughput manner relative to costly animal
studies. (7) Last but not least, these assays allow us to
study and manipulate human cellular responses in
physiological context. For a brief review of the results
obtained with these models and studies in rodents
cells in 3D cultures and in vivo
, see Bissell et al.
For a history of development of 3D cultures in general
see Schmeichel and Bissell (2003). For a review of more
complex organotypic cultures, see Gudjonsson et al.
II. MATERIALS AND
Primary breast luminal cells from human reduction
mammoplasty (Petersen et al.
, 1992) as well as human
mammary epithelial cell lines have been successfully
cultured utilizing the 3D BM assay. These include the
The HMT3522 progression series (Briand et al.
1987) consists of immortal human mammary epithelial
cells originally isolated from fibrocystic breast
tissue and includes the phenotypically normal S1
cells, as well as their tumorigenic derivative T4-2
cells, which were selected for their ability to grow in
the absence of EGF (Briand et al.
, 1996; Weaver et al.
This article focuses mainly on the HMT3522 series
but provides examples of the morphology of a few
other nonmalignant cell lines, such as 184B5 (Walen
and Stampfer, 1989); for a description of these series,
see http://www.lbl.gov/~mrgs/review.html and
MCF10A (Soule et al.
, 1990). For further information
and culture conditions, see http://www.atcc.org
/ATCC Number: CRL-10317.
A. Culture Media Composition
Media for the HMT3522 progression series of
human mammary epithelial cells are composed of
DMEM/F12 media (GibcoBRL Cat. No. 12400-024)
supplemented with insulin (Sigma Cat. No. 1-6634),
human transferrin (Sigma Cat. No. T-2252), sodium
selenite (Collaborative Research Cat. No. 40201), estradiol
(Sigma Cat. No. E-2758), hydrocortisone (Sigma
Cat. No. H-0888), prolactin (Sigma Cat. No. L-6520),
and epidermal growth factor (EGF) (Roche Cat. No.
855731) as described in Briand et al.
Blaschke et al.
(1994). Concentrations and stability are
outlined in Table I.
B. Additional Reagents Required for Culture
and Manipulation in Three-Dimensional
Vitrogen (collagen I, Cohesion Technologies) can be
made from rat tail collagen, phosphate-buffered saline
(PBS) (any vendor), 0.25% trypsin-EDTA (any vendor),
soybean trypsin inhibitor (Sigma Cat. No. T6522),
trypan blue (Sigma Cat. No. T8154), growth factor
reduced Matrigel (Becton-Dickenson-Collaborative
Research Cat. No. 354230), AIIB2, blocking antibody
against 131 integrin (Sierra BioSource Inc. Cat. No.
SB959), LY294002 (Cell Signaling Technologies Cat.
No. 9901), Mab225 (Oncogene Research Cat. No.
GR13L), PP2 (Calbiochem Cat. No. 529573), PD98059
(Calbiochem Cat. No. 513000) and tyrphostin AG1478
(Calbiochem Cat. No. 658552). All other equipment
and supplies can be purchased from a variety of
C. Minimal Equipment Required for Cell
Hemacytometer, low-speed centrifuge, inverted
light microscope equipped with 4, 10, 20, and 40× objectives, pipetman p20, p200, pl000, pipette aid, biological
safety cabinet, 37°C, 5% CO2
incubator, air-tight plastic container, 37~ water bath.
D. General Tissue Culture Supplies
Tissue culture plasticware including T75 flasks, 4-,
6-, 24-, 48-, and 96-well dishes, 5-, 10-, and 25-ml individually
wrapped sterile pipettes, 15- and 50-ml sterile
conical centrifuge tubes, sterile 1- to 200- and 500- to
1000-µl pipette tips, and sterile 9-in. Pasteur pipettes
and cell lifters.
Successful growth of these cells requires special
attention to the media, confluency, trypsinization, and
feeding regime in order to maintain healthy cells
capable of undergoing differentiation. Insufficient
attention to any of the aforementioned parameters will
quickly result in cells acquiring a fibroblast-like morphology
and concomitant failure to undergo 3D organization
when cultured in IrBM.
A. Cell Maintenance
1. Preparation of Collagen-Coated Tissue Culture
2. Passage of HMT3522 Cells
- Make a working solution of collagen I [~65µg/ml]
by the addition of 1 ml Vitrogen (collagen I) to 44 ml
of cold l× PBS.
- Add 3ml of collagen solution for every 25-cm2 surface area of flask; tilt flask to make sure entire
surface is coated.
- Store in an air-tight container, on even surface at
4°C, a minimum of overnight and a maximum of 3
- Before use, aspirate liquid in the flask, wash once
with 2ml DMEM/F12 per 25cm2 and add prewarmed
Change media every 2-3 days; it is important to
ensure that fresh media be added 1 day before splitting.
HMT3522 S1 cells should be passaged once the
2D colonies form rounded islands with the flask being
approximately 75% confluent, as shown in Fig. 1A.
This is usually occurs 8 to 10 days after the initial
Passage HMT3522 T4-2 cells when the flask reaches
75% confluency, as shown in Fig. lB. This usually
occurs 3 to 5 days after initial plating. Maintain T4-2
cells on Vitrogen (collagen I) or rat tail collagen-coated
|FIGURE 1 The morphology of S1 and T4-2 cultured on tissue culture plastic. (A) S1 cells, passage 57, at
day 10 after plating. The colonies are essentially round with smooth edges and with the inner cells somewhat
compacted. Cells at the edge should take on a polar-like appearance indicated by the arrows. (B) T4-2,
passage 37, at day 5 after plating. Cells are larger than $1, the colonies are irregular, and cells at the edges
rarely show any organization. Bar: 25 µm.
Perform the following steps in a tissue culture hood.
3. Three-Dimensional BM Assay Embedded in
I rBM/EHS/(Ma trige 1)
- Aspirate medium.
- Rinse cells in 0.5 to 1 ml of prewarmed 0.25%
trypsin-EDTA for every 25 cm2 of surface area.
- Add 0.35ml of 0.25% trypsin for every 25 cm2 of
surface area and cover cells by rotating the flask gently.
- Place flask in 37°C incubator for 3 min.
- Remove flask from incubator and check under
the microscope to see if the cells have detached.
- If cells are still adherent, redistribute the trypsin
and return the flask to the 37°C incubator for 1
min. Repeat until cells are detached.
- Once the majority of the cells are detached,
knock the side of the flask gently; add -3 ml of
prewarmed DMEM/F12 and 30µl of soybean
trypsin inhibitor per 25 cm2.
- Gently pipette cells up and down three to five
times to dissociate cell aggregates and transfer to a
15-ml conical centrifuge tube.
- Pellet the cells by spinning at ~115g for 5 min.
- Aspirate media and resuspend the cell pellets in
their appropriate growth media (~3.25 ml per 25 cm2 of
surface area of their original flask).
- Determine the cell concentration by removing a
95-µl aliquot and placing into a 1.5-ml Eppendorf tube,
add 5µl of 0.4% trypan blue, mix gently with a pipetman,
load onto a hemacytometer, count on an inverted
microscope, and determine the number of viable
(nonblue) cells per milliliter.
- HMT 3522 S1 cells at a density of 2 × 104 cells/cm2 on tissue culture plastic.
- HMT 3522 T4-2 cells at a density of 1 × 104 cells/cm2 on collagen I-coated flasks.
Single phenotypically normal mammary epithelial
cells embedded into lrBM will undergo several rounds
of cell division, withdraw from cell cycle between 6
and 8 days (depending on the cell type), organize into
polarized structures, and form acini-like structures,
including a central lumen. Malignant cells, however,
continue to proliferate and form disorganized, tumorlike
structures (Fig. 2). Starting on day 4 but clearly by
day 10, one can distinguish nonmalignant from malignant
cells. Immunohistochemistry analysis for the Ki67
antigen on day 10 shows that ~60% of the malignant
cells are still proliferating, whereas only <10% of the
nonmalignant remain in the cell cycle.
|FIGURE 2 Morphology, size, and proliferation rate of S1 and T4-2 and T4-2 reverted cells embedded in
3D BM. (A) Schematic representations of the morphology of S1, T4-2, and T4-2 cells treated with signaling
inhibitors as a function of time. (B) The percentage of Ki67 positive cells for S1, T4-2, and T4-2 cells treated
with tyrophostin AG1478 from days 2 to 10. (C) The number of cells/acini for S1, T4-2, and T4-2 cells treated
with tyrophostin AG1478 from days 2 to 10.
The following volumes and concentrations given
are appropriate for a 35-mm tissue culture dish (9.6-
surface area). The volumes and concentrations
must be adjusted to correct for the area of the plate
used in the analysis. IrBM should be stored at -80°C. Thaw immersed in ice
at 4°C overnight before use.
Once thawed it should not be refrozen
but can be kept in ice
at 4°C for more than 1 month. Matrigel lots are
tested before use and batches are purchased for experimental
use. Alternatively, the basement membrane
mixture (EHS) can be made from the Engelbreth-Holm-Swarm mouse tumor described in Kleinman et al.
(1982); see Section III,B details.
: EHS or Matrigel should always be kept on ice
as it will polymerize quickly at room temperature.
4. Three-Dimensional BM Assay on Top of EHS/
- Chill plates on ice inside a tissue culture hood.
- Add 120µl of EHS per 35-mm dish spread in the
center of the dish without introducing bubbles, spread
EHS evenly across the surface of the dish using a
sterile cell lifter or the back of a sterile pipette tip
(make sure the entire surface is covered, including the
edges), and place in a 37°C incubator for a minimum
of 15 min to allow the EHS to polymerize. (Do not let
sit more than 1h in incubator as it will begin to dry
- While the EHS is polymerizing, trypinsize and
count the cells as described earlier.
- Place an aliquot containing the appropriate
number of cells into a sterile centrifuge tube: 1.0 × 106 cells S1 (and other nontumorgenic cell lines) and 0.7× 106 cells T4-2 (and other tumorgenic cell lines).
- Centrifuge for 5 min at ~115g to pellet the cells,
aspirate media, and resuspend the pellet by flicking
- Place the cell pellet on ice, add 1.2ml EHS, and
carefully pipette mixture up and down to distribute
cells evenly but not introduce bubbles into the EHS.
- Transfer to a precoated 35-mm dish.
- Place in 37°C incubator for ~30 min until the EHS
- Once a gel is formed, add 1.5 to 2.0ml of the
appropriate media to the dish.
- Culture the cells for 10 days, changing media
every 2 to 3 days.
Figures 3A-3D show cells cultured in Matrigel for
The culture of mammary epithelial cells, developed
previously in our laboratory for functional studies of
mouse cells (Barcellos-Hoff et al.
, 1989; Roskelley et al.
1994), has now been adopted to human cells as well.
Making 3D BM cultures on top of EHS as opposed to
inside the gel has some advantages and a few drawbacks.
The proliferation and morphological differences
between nonmalignant and malignant cells can be distinguished
by 4 days in culture as opposed to the 8-10
days required for embedded cultures. The cell colonies
can readily be imaged utilizing a live cell imager,
allowing one to follow a single cell to an acini-like
structure. Furthermore, cell colonies can be harvested
easily by scraping the culture gently with a pipette tip
and depositing the isolate on a glass slide for analysis by immunohistochemistry. The advantage of this
method of harvest is that the remaining culture can
be harvested for DNA, RNA, or protein. However,
neither the morphology nor the growth rate of the
acini is as tightly controlled as the embedded cultures.
An additional disadvantage of the on-top
culture is that
the amount of material collected per plate is less than
the embedded cultures, as plating of the cells is
restricted to one plane. Therefore, there are significantly less cells per millimeter dish to be harvested
compared to the embedded assay.
The following volumes and concentrations are
appropriate for a 35-mm tissue culture dish (9.6-cm2
surface area). The volumes and concentrations must be
adjusted to correct for the area of the plate used in the
Before splitting the cells, prepare the EHS-coated
- Chill plates on ice in a tissue culture hood.
- Add 0.5 ml to the center of the dish and spread
EHS evenly without creating bubbles as described
- Place in 37°C incubator for ~30min until the EHS
- Trypsinize cells as described previously.
- Place an aliquot containing the appropriate number of cells into a sterile centrifuge tube: 0.3 × 106 cells S1 (and other nontumorgenic cell lines) and 0.2× 106 cells T4-2 (and other tumorgenic cell lines).
- Centrifuge for 5 min at ~115g to pellet the cells,
aspirate media, and resuspend the pellet by flicking
- Resuspend the cells in half of the total volume of
the appropriate media for the cells (1 ml for 35mm)
according to the size of the plate (see later).
- Plate the cells on top of the polymerized EHScoated
dishes, let sit in hood for 5 min, and then check
under a microscope to see if cells are distributed
evenly. If not, move plate on a flat surface, back to front
and then side to side, to distribute and let sit for 5 min.
- Return plates to the incubator for-30min until
the cells have adhered.
- Prepare media plus 10% EHS on ice.
- Add the appropriate amount of media with 10%
EHS to the cells to achieve a final concentration of 5%
- Culture the cells for 4-6 days, changing media
every 2-3 days.
|FIGURE 3 Morphologies of $1 (A), T4-2 (B), 184B5 (C), and MCF10 (D) cells embedded in 3D BM (all cells
were cultured in Matrigel for 10 days) and S1 (E) and T4-2 (F) cells cultured on top (cells were cultured on
top of Matrigel for 4 days). Photographs were taken using a phase-contrast microscope. Bar: 25 µm.
5. Reversion Assay
The reversion assay can determine the ability of
tumor cells to become phenotypically normal or to
undergo apoptosis as a function of treatments. In addition,
the differential response of tumorigenic and nonmalignant
cells to a wide variety of perturbations can
be analyzed. Analyses can be performed both on cells
cultured embedded in or on top of EHS. This assay
can be used as a screen for potential therapeutics
by manipulating various genes, signaling pathways,
and/or protein modifications and for determining
their role in the establishment or maintenance of the
tumorigenic phenotype. Cancer cells, cultured as
described earlier, can be treated with a wide variety
of agents, including inhibitory or stimulatory
antibodies to integrins or growth factor receptors,
small molecule inhibitors to different signaling
pathways, gene delivery via transfection with
expression vectors or transduction with virus overexpressing
genes of interest, and RNAi for specific gene
Quantitative end points include morphology, proliferation
index, degree of polarity, and level of expression
of genes of interest using RT-PCR, Northern, or
Western analysis, as well as immunofluorescence.
Table II details a variety of molecules that have been
shown to revert T4-2 cells. A typical reversion morphology
of T4-2 cells is illustrated in Fig. 4.
|FIGURE 4 Morphological and immunofluorescence analyses of S1, T4-2, and T4-2 treated with LY 294002
or tyrphostin AG1478. (A) Cells were embedded in Matrigel and cultured for 10 days. (B) Cells were cultured
for 4 days on top of Matrigel. S1, T4-2, and T4-2Rev cells were treated with 0.1 mM AG1478 tyrphostin. Photographs
were taken using a phase-contrast microscope. A & B Bars: 25 mm. (C) Structures isolated from 3D
BM after 10 days in culture were stained with the basal marker α6- integrin, the apical marker ZO-1, and the
cytoplasmic marker F-actin. S1, T4-2, and T4-2Rev cells were treated with 10mM LY 294002. Photographs
were taken using a fluorescence microscope. Bars: 10mm.
6. Release of Cellular Structures from 3D BM
For molecular analysis of cell extracts, it is important
to remove them from the gel. Apart from scraping
the colonies as described earlier, which will still have
minor EHS contamination, intact cellular structures
can be released from EHS utilizing chelating agents, a
procedure described in Weaver et al.
(1997). A modified
version is described here. Note that the solutions
must be cold and the cells kept on ice for the entire
harvest period. The maximum harvest should be 1 h as
the cell-cell junctions will begin to come apart if left in
these solutions for too long.
Perform the following steps on the day of harvest.
- Wash cultures twice with cold PBS without Ca2+ or Mg2+ plus 0.005 M EDTA.
- Scrape cultures into 3 ml cold PBS without Ca2+ or Mg2+ plus 0.005 M EDTA/25-cm2 surface area, transfer
into an appropriate size centrifuge tube, wash the
plates twice with 3 ml each cold PBS, and bring final
volume to 10 ml.
- Incubate cells on ice for ~45 min, gently inverting
the tube two or three times every 10 min.
- Cell release from 3D BM is complete when the
cellular structures begin to settle at the bottom of the
centrifuge tube and the supernatant does not contain
floating gel or flocculent substances.
- Centrifuge gently for 2-4min at 115g until a
loose pellet is obtained (the cells are very fragile at this
point; therefore, centrifuging too hard or long will
result in cell lysis).
- Aspirate the supernatant, leaving a very small
amount of liquid above the pellet.
- For immunohistochemistry or immunofluorescence,
smear a small aliquot gently across a small
area of a glass slide and fix, using the appropriate
fixative for antibody of interest and store at-
20°C until use.
- For RNA isolation, lyse an aliquot in lysis buffer
of choice and store at -80°C.
- For Western or immunoprecipitation analyses,
lyse aliquots in appropriate buffer and store at
: Trizol or Tripure can be used to isolate RNA
and DNA directly from the cultures without release of
the cells from the gel.
B. Additional Procedures
1. Criteria for Purchase of Appropriate Matrigel
Test lots of Matrigel and choose those that are
low in epidermal growth factor (EGF), low endotoxin,
which have a protein concentration close to 10mg/
Test the cells of interest by always comparing old
and new lots side by side using the 3D BM-embedded
and on top
culture conditions as described earlier. Also
test the ability of malignant cells of interest to revert in
the presence of tyrphostin AG1478 or known reverting
2. Preparation of EHS Matrix from EHS Tumors
- On the final day of culture measure the colony
sizes using an inverted microscope equipped with an
eyepiece reticule. Eighty percent or greater of the
colonies should be within the following diameters: 24
µm S1, >74 µm T4-2, and 25-32 µm T4rev.
- Isolate colonies from the 3D BM as described
previously to determine:
- by immunofluorescence
- cell proliferation index by determining the
percentage of positive Ki67 cells
- degree of polarity by localization of molecules
such as α6-integrin, E-cadherin, and cortical
- by Western blot (for S1 and T4-2)
- relative levels of β1 integrin
- relative levels of EGF receptor
The EHS tumor is grown in C57 black mice. Typical
propagation of the tumor starts with one-third of a
fresh tumor, which is enough to inject 10 mice. The
tumor sample is minced with a scalpel blade sussuspended
into 2 ml PBS and is then further disassociated
by pushing it through an 18-gauge needle followed by
a 20-gauge needle. This tumor slurry is then injected
intramuscularly (IM) into the hind limb (0.2ml/limb)
of mice. Tumors are allowed to grow for 4 weeks. For
the last 2 weeks of tumor growth, 0.1% β-aminopropionitrile
fumarate (BAPN) is added to the drinking
water to prevent the collagen from cross-linking. The
tumors should be harvested, weighed, frozen in liquid
nitrogen, and stored at-80°C until use. For further
information, see Kleinman et al.
The following buffers are needed for isolation.
Buffer 1: 3.4M
Tris-Cl, pH 7.4,
EDTA, and 2mM N
(make up 100× stock in H2
O fresh for each other
Buffer 2: 0.2M
Tris-Cl, pH 7.4, 4rmM
NEM (make up 100× stock in H2
O fresh for each preparation), and 2M
Buffer 3: 0.15M
Tris-Cl, pH 7.4, 4mM
EDTA, and 2 mM
NEM (make up 100× stock in H2
O fresh for each preparation). Sterilize
All solutions should be at 4°C and kept on ice
- Distribute 10-15 g of frozen tumor tissue (stored
previously at -80°C) into two 50-ml tubes.
- Add approximately 2ml buffer 1 for each gram
of tissue and homogenize (add more buffer as needed
until all tissue is homogenized evenly).
- Transfer homogenate to an appropriate ultracentrifuge
tubes, balance tubes using buffer 1, and spin
for 20min at 4°C at 83472 RCF (ave) (corresponds to
26,000rpm when using a SW 41 rotor).
- Decant supernantant, add 1-2ml fresh buffer 1,
rehomogenize, transfer to fresh ultracentrifuge tubes,
balance with buffer 1, and spin again as just described;
repeat these steps so that the tissue has been homogenized
and spun for a total of three times.
- Resuspend the pellet in buffer 2 as follows: add
1 ml/tube, homogenize briefly to get pellet into suspension,
add buffer 2 until equivalent to 1.8ml/g of
original tumor weight, transfer suspension into a
beaker with a stir bar, cover well, and stir overnight in
a cold room.
- Transfer liquid to ultracentrifuge tubes and
spin as described earlier (try to add the minimum
amount of additional buffer to balance). Save the
- Load supernatant into medium-sized dialysis
tubing. Dialyze in cold room against 1 liter of buffer 3
for 1.5 to 2 days with three changes of buffer.
- Dialyze in cold room against the media of choice
- Store on ice in thermos at 4°C if used within 2
months. Otherwise, freeze aliquots at -80°C and thaw
on ice before use.
Using this assay system, one can screen a wide
variety of breast tumor cell lines and assess their
ability to revert or die when treated with various signaling
inhibitors and/or potential therapeutics, as
outlined in Wang et al.
- Use healthy cells that are no more than 75%
- Make sure the cells have been dispersed into single
cells and that you do not introduce bubbles into the
lrBM for 3D BM-embedded cultures. Cell clumps
will make it hard to interpret morphological characteristics
at the end of the assay.
- Feed cells every 2-3 days with fresh media when
appropriate, include test compound.
Always include a cell line with a known positive
response when performing the reversion assay.
Work from the authors' laboratory was supported
by the United States Department of Energy, Office of
Biological and Environmental Research (DE-AC03
SF0098 to M.J.B.). Additional funding was contributed
by the Department of Defense Breast Cancer Research
Program (#DAMD17-02-1-0438 to M.J.B.) and the
National Cancer Institute (CA64786-02 to M.J.B.,
and CA57621 to Zena Werb and M.J.B). The authors
thank Paraic Kenny for editorial comments.
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