(a) Human Lung Carcinoma. Outgrowth from primary
Astroglial cells from an anaplastic
explant from squamous cell carcinoma of human lung,
MOG-L-DAN. Giemsa stained. 10x objective.
(c) Human Cervical Intraepithelial Neoplasia (CIN).
Primary culture from cervical biopsy subcultured on to
3T3 feeder layer of which a few degenerating cells
remain, top right. Giemsa stained. 10x objective.
(courtesy of M. G. Freshney.)
Plate 1. Primary Culture, Human.
(b) Human glioma.
astrocytoma. Giemsa. 40x objective.
(d) Human Fibrosarcoma. Primary culture subcultured on to
fetal intestinal feeder layer. The fibrosarcoma cells are
spindle shaped and the feeder layer polygonal. Giemsa
stained. 10x objective.
 (b) Primary
Explant. Mouse
squamous cell
carcinoma.
Phase contrast;
4x objective.

(c) Collagenase
Digest. Human
colon carcinoma.
Bright field; 4x
objective.

(a) Mouse Kidney Tubules. Disaggregation of newborn

mouse kidney by cold trypsin method. Connective tissue has
dissociated, but fragments of tubules and glomeruli remain
intact. Also seen with collagenase digestion. Normal bright
field illumination; 4x objective.

(d) Outgrowth from Kidney Tubules. Outgrowth of cells (e) Newborn Rat Kidney. Primary culture from trypsinized
from attached fragments following disaggregation by (cold method) newborn rat kidney. Giemsa stained; 10x
cold trysin method. Phase contrast; 10x objective. objective. (Courtesy of M. G. Freshney).

Plate 2. Primary Culture, Explant, Cold Trypsin, and Collagenase.

(a) Chick Embryo Lung. Primary culture of 13-day chick (b) Chick Embryo Liver. Details as for (a).
embryo lung, 48 h after disaggregation by the cold trypsin
method. Light Giemsa stain and phase contrast; 10x objective.

(c) Chick Embryo Thigh Muscle. Culture details as for (d) Chick Embryo Kidney. Culture details as for (a).
(a). Note multinucleate myotube (red arrow) resulting from Giemsa stain, bright field; 10x objective.
fusion of myocytes (small dark-stained spindles). Giemsa
stain, bright field; 10x objective.

Plate 3. Primary Culture, Chick Embryo Organ Rudiments.

(a) Newly subcultured monolayer. NRK cells 24 h after (b) Entering log phase. NRK cells 48 h after subculture. Phase
subculture. Phase contrast; 10x objective. contrast; 10x objective.

(c) Mid-log phase cells. NRK cells 3 days after subculture; (d) Late log phase cells. NRK cells 7 days after subculture and
ready for medium change. Phase contrast; 10x objective. ready for subculture again. Phase contrast; 10x objective.

Plate 4. Phases of the Growth Cycle.

(a) NRK Monolayer before Trypsinization. Phase contrast; 20x objective. (b) NRK Monolayer after D-PBSA/EDTA Prewash. Phase contrast; 20x
objective.

(c) NRK Monolayer Immediately after Trypsin Removal. Phase contrast; (d) NRK Monolayer 1 min after Trypsin Removal. Phase contrast; 20x
20x objective. objective.

(e) NRK Monolayer 5 min after Trypsin Removal. Phase contrast; 20x (f) Fully Disaggregated Monolayer. 10 min after removal of trypsin and
objective. ready for dispersing and counting. Phase contrast; 20x objective.

Plate 5. Subculture by Trypsinization.

 5 mm

(a) HeLa Clones. HeLa-S3 cloned by dilution cloning and stained with (b) NRK Clone. Small clone of NRK cells, cloned by dilution cloning.
Giemsa after 3 weeks. Scale bar 5 mm. Phase contrast, 4x objective.

(c) Breast Carcinoma, JUW, Cloned on Plastic. Microphotograph of (d) Breast Carcinoma on Feeder Layer. Microphotograph of epithelial
secondary culture from human breast carcinoma, 4000 cells/cm2, colony from human breast carcinoma cells, 400 cells/cm2, growing on
growing on plastic. Mainly fibroblasts. Giemsa stain. 10x objective. confluent feeder layer of FHS74Int human fetal intestinal cells. Giemsa
stain. 10x objective.

10 mm

(e) Effect of Serum on Plating Efficiency. Mv1Lu cells, transfected

with myc oncogene, plated with 500 cells per 6-cm Petri dish, and
fixed and stained 2 weeks later. 10% FBS (left), 20% FBS (right).
Scale bar 10 mm. (Courtesy of M.Z. Khan.)

Plate 6. Cell Cloning.

 10 mm

(a) Clone of Continuous Glioma Cell Line, MOG-G-CCM. Elliptical (d) Cloned Culture of Early Passage Cell Line. Human non-small cell
and spindle-shaped morphology. Giemsa stained. 10x objective. lung carcinoma (NSCLC). Scale bar 10 mm.

(b) Different Clone of Same Continuous Glioma Cell Line,

MOG-G-CCM. Epithelioid morphology. Giemsa stained. 10x
objective. (e) Clones from Human NSCLC. Detail from (d). Giemsa stained.
4x objective.

(c) Third Clone of Continuous Glioma Cell Line, MOG-G-CCM.

Squamous epithelioid morphology. Same magnification as (a) and
(b). Giemsa stained. 10x objective.
(f) Cloned Culture of A2780. Continuous cell line from human ovarian
carcinoma. Giemsa stained. 4x objective.

Plate 7. Cell Cloning, Morphological Diversity.

 (a) (b)

(a) Normal Human Fetal Lung Fibroblasts. Subconfluent normal fetal (b) Confluent Fibroblasts. Dense culture of normal fetal
human lung fibroblasts. Giemsa stained; 10x objective. human lung fibroblasts. Phase contrast; 10x objective.

(c) (d)

(c) Human Umbilical Cord Endothelium. Cell line

from collagenase outwash of human umbilical vein.
Giemsa stained. 20x objective.

(e)

(d) Human Melanocytes. Secondary culture of TPA-treated

epidermal melanocytes derived from African-American
newborn foreskin. Note the dendritic morphology of the cells
and their slender spindle shape. In contrast to the
melanocytes derived from Caucasian newborn foreskin,
these melanocytes display a high level of melanin granules
(seen via phase contrast, x320). (Courtesy of H.-Y. Park.)

(e) View of Blood Vessels in Human Umbilical Cord.

Preparation for enzymatic isolation of endothelial cells from
umbilical vein (see Protocol 22.18A). Black arrows, umbilical
arteries. White arrow, umbilical vein with Luer adaptor inserted.
(Courtesy of Charlotte Lawson; see also Fig. 22.3.)

Plate 8. Finite Cell Lines and Cannulation of Human Umbilical Cord.

(a) HeLa-S3. Human cervical carcinoma. Phase (b) A549. Human lung adenocarcinoma. Giemsa
contrast. 40x objective. stained. 10x objective.

(c) MOG-G-UVW. Human glioma. Giemsa stained. (d) Caco-2. Human colorectal carcinoma showing dome formation.
10x objective. This cell line should be subcultured before it reaches this stage and
while it is still subconfluent. Phase contrast; 10x objective.

Plate 9. Continuous Cell Lines from Human Tumors.

(a) CHO-K1. Clone of CHO, continuous cell line from Chinese hamster (b) Swiss 3T3. Fibroblast-like cells from Swiss mouse embryo.
ovary. Phase contrast. Giemsa stained.

2 mm

(c) BHK-21 Clone 13. Baby hamster kidney fibroblasts after (d) BHK-21 Clone 13. Baby
confluence showing typical swirling pattern formed by parallel hamster kidney fibroblasts
arrays of cells. Giemsa stained. approaching confluence and
assuming parallel arrays. Giemsa
stained.
(e) High-Density BHK-21 C13. Baby
hamster kidney fibroblasts
postconfluent and forming a second
monolayer of more densely stained
cells. Giemsa stained.

200 µm

(f) MDCK. Madin Darby canine kidney. Epithelial-like (g) Mv1Lu. Mink Lung Epithelial Cell Line. Stained by immunoperoxidase
cell line from dog kidney. for cytokeratin (AE3 primary antibody; Photo courtesy of M.Z. Khan.)

Plate 10. Continuous Cell Lines from Normal, Nonhuman Animals.

(a) Normal Human Keratinocytes on 3T3 Feeder Layer. Keratinocyte (b) Human Glioma. MOG-G-CCM cells stained for GFAP by
pancytokeratin stained green, and vimentin in 3T3 cells, red. (Courtesy of immunoperoxidase.
Hans-Jurgen Stark.)

(c) Breast Stromal Fibroblasts. Immunoperoxidase stained for vimentin (brown). (d) Human Umbilical Vein Endothelial cells. Factor VIII
(Courtesy of Valerie Speris.) granular staining by immunoperoxidase.

Plate 11. Immunostaining.

 (b) Dome Forming in Monolayer of WIL Lung Adenocarcinoma.
Mosaic of CEA-positive and CEA-negative cells. Focused on monolayer.
(a) Dome Forming in Monolayer of Wil Lung Adenocarcinoma.
Anti-CEA immonoperoxidase stained. 40x objective.
Mosaic of CEA-positive and CEA-negative cells. Focused on top of
dome. Anti-CEA immonoperoxidase stained. 40x objective.

1 mm

(c) A549 Lung Adenocarcioma Cells Growing on Matrigel. (d) A549 Lung Adenocarcinoma Cells. Growing in non–tissue-culture grade
Upper, 4x objective, lower 10x; bright field, unstained. Petri dish, with lung fibroblasts growing on a coverslip in center of dish. 10x
(Courtesy of Jane Sinclair.) objective; bright field.

Plate 12. Morphological Differentiation in Epithelial Cells.

 (a)
(a, b) Hemoglobin in Friend Cells. Benzidine staining of hemoglobin in
control Friend erythroleukemia cells. (a) control, (b) with 2% DMSO.
(Courtesy of David Conkie.)
(b)

(c)
(c, d) In situ Labeling of Friend Cells. Effect of DMSO on induction of
mRNA for globin. (c) control, (d) induced with 2% DMSO. (Courtesy of
David Conkie.)
(d)

(e) (f)
(e, f) Morphological Differentiation in Human Glial Cells. (e) Undifferentiated glial cells from normal human brain. (f ) Morphological
differentiation induced in glial cells from normal human brain by glia maturation factor. Giemsa stained.

Plate 13. Differentiation in Friend Cells and Human Glia.

(a) Contact Inhibition. Late log-phase cultures of BHK21-C13
Cells tend to assume a parallel orientation and will not overgrow
each other. Phase contrast. 10x objective.
20 mm
20 mm
(c) Focus Formation in 3T3 Cells. A monolayer of Sw-3T3
cells, left at confluence for 3 weeks, showed foci of
transformed cells escaping contact inhibition. (top), Whole
flask; (bottom) 4x objective. Giemsa stained.
Plate 14. Properties of Transformed Cells.
(b) Loss of Contact Inhibition. Polyoma-transformed clone
BHK21-PyY cells that show no recognition of each other and
grow randomly over each other. Phase contrast. 10x objective.
(d) [3H]-thymidine Incorporation in Mv1Lu mink Lung Cells. Mink
lung cells, Mv1Lu, were labeled with [3H]-TdR for 1 h, fixed, and
coated with autoradiographic emulsion (see Protocol 27.3), and
stained with Giemsa. Mv1 (top) is the control cell line Mv1Lu, M1
(middle) was Mv1Lu transfected with the myc oncogene, and T1
(bottom) with mutant ras. T1 cells were tumorigenic and had a
statistically significant (p<0.001) increase in labeling index
compared to Mv1Lu, also seen with bromodeoxyuridine labeling
[Khan et al., 1991]. (Courtesy of M. Z. Khan.)
 (a) (b)
(a, b) Infiltration of Normal Monolayer. (a) Contact inhibited confluent monolayer of normal human fetal instestinal cell line FHS74Int.
(b) Human glioma cell Line WLY infiltrating FHS74Int. Normal glial cells do not infiltrate this confluent monolayer 10x objective; Giemsa
stained.

(e-i)

(c) (d)
(c, d) Non–Small Cell Carcinoma Cell Line Infiltrating Normal
Fibroblasts. (c) Phase contrast. (d) Indirect immunofluorescence
for cytokeratin revealing the tumor cells migrating in parallel with
the fibroblasts. 40x objective.
(e) In vitro
Angiogenesis Assay.
(e-ii)
Endothelial cells grown on Cytodex beads induced to form tubes; (e-i)
Endothelial cells forming a monolayer on the surface of the Cytodex
beads (control) (e-ii) Endothelial cells grown on beads induced to form
tubes by treatment with 10 ng/mL FGF. Stained with DAPI (see
Protocol 17.5).

(f) (g)
(f, g) Induction of Angiogenesis. A crude extract was placed on the chorioallantoic membrane at 10 days of incubation, and the membrane
was removed 2 weeks later. (f) Normal glial cell extract. (g) Crude extract of Walker 256 carcinoma cells. (Courtesy of Margaret Frame.)

Plate 15. More Properties of Transformed Cells.

(a) Contaminated Flask. Reduction in pH (b) Floculated Contamination. Bacterial, but no drop in pH.
and cloudiness of medium.

Control Negative Positive

test control

(c) Contaminated Medium Bottle. Cloudy (d) Samples of Broth. L Broth with test samples of medium. Control (left) clear,
with no drop in pH; yeast. Negative test (center), and positive control (right).

(e) Mycoplasma, Low Power. Mycoplasma (f) Mycoplasma, High Power. Hoechst-stained mycoplasma infected cells under
infected culture as revealed by Hoechst 33258 100x oil-immersion objective.
staining. 40x water-immersion objective.

Plate 16. Examples of Contamination.

(a) Dye Exclusion, Naphthalene Black.
Hemocytometer slide 200-mm square with viable
(unstained) and nonviable (blue stained) cells. 40x
objective.

Plate 17. Viability and Cytotoxicity.

 (i)

(a) Transfected Mosaic Spheroids Derived from Human

Glioma Cell Line MOG-G-UVW. The Spheroids, ranging (ii)
in size from 100 to 500 µm diameter, are composed of
mixtures of cells transfected with the GFP gene (green)
and cells transfected with the NAT gene (red). (Courtesy
of Marie Boyd and Rob Mairs; see Section 25.3.3.)

(iii)

(c) Microcarriers. Vero cells growing on microcarriers.

(Courtesy of MP Biomedicals.)

(b) Alginate Encapsulation. Light microscopic

images of cells encapsulated in alginate after
2 hours (i), 3 weeks (ii) and 4 months (iii) in vitro.
Within the alginate beads both cell death and cell
proliferation will occur, and for many cell lines
multicellular spheroids will form inside the beads.
(Courtesy of Tracy-Ann Read and Rolf Bjerkvig;
see Section 25.3.5.)

(d) Hexagonal Microcarriers. Nunc Microhex Beads.

(Courtesy of Nunc.)

Plate 18. Spheroids, Encapsulation, and Microcarriers.

 (a) (b)
(b) Human Fetal Lung Epithelial Cells. Cells growing on filter with
human fetal lung fibroblasts on other side of filter. 4x objective.
Giemsa stained.

(c) (d)
(c, d) Lung Cell Coculture. Fetal lung epithelial cells and fibroblasts as in (b). (c) Focused on cells above filter. (d) Focused on cells below filter.
40x objective. Giemsa stained.

(e)
(e) Section of Coculture. Section through filter with A549 cells and
fibroblasts. Both cell types were seeded on top, but fibroblasts migrated
through first, though some remained on the top. Three cells are seen
travelling through the pores. 40x objective; H&E stain.
(f)
(f) Chick Embryo Lung. High-density primary culture of cold
trypsin disaggregated 13d embryonic chick lung. 72 h after seeding at
>1x106 cells/mL. 40x objective. Giemsa stained.

Plate 19. Organotypic Culture in Filter Wells.

(a) Keratinocyte and Dermal Fibroblast Cocultures. Organotypic (b) Organotypic Cultures Implanted In vivo. Organotypic cocultures
cocultures after 2 weeks in vitro. 40x objective. H&E stain. in vivo 3 weeks after transplantation onto the nude mouse. 40x
objective. H&E-stain.

(e) Involucrin and Collagen in Organotypic Cocultures at 2 weeks. (f) Involucrin and Collagen in Organotypic Cocultures at 3 weeks.
Keratinocyte-fibroblast cocultures with collagen gel stained for Keratinocyte-fibroblast cocultures with collagen gel stained for
involucrin (green) and the basement membrane component collagen involucrin (green) and the basement membrane component collagen VII
VII (red). 40x objective. (red). 40x objective.

Plate 20. Organotypic Culture of Skin. (Courtesy of Hans-Jürgen Stark)

(a) Exposed to Control Solution for 10 min. (b) Exposed to 1% SDS for 10 min.

(c) Control Solution for 20 min. (d) 1% SDS for 20 min.

(e) Control Solution for 60 min. (f) 1% SDS for 60 min.

(See also Figs. 21.10, 25.7) (Examples, courtesy of SkinEthic.)

Plate 21. In vitro Toxicity in Organotypic Model.

 (a) Sterilizing Bottles. These bottles were
(a) autoclaved with Thermalog sterility indicators
inside. Thermalog turns blue with high temperature
and steam, and the blue area moves along the strip
with time at the required sterilization conditions.
The cap on the leftmost bottle was tight, and each
succeeding cap was gradually slacker, until, finally,
no cap was used on the bottle furthest to the right.
The leftmost bottle is not sterile because no steam
entered it. The second bottle is not sterile either
because the liner drew back onto the neck and
sealed it. The next three bottles are all sterile, but
the brown stain on the indicator shows that there
was fluid in them at the end of the cycle. Only the
bottle at the far right is sterile and dry.
The glass tube indicators (Browne’s Tubes),
which are temperature sensitive only, all indicated
that the bottles were sterile.

(b) pH Standards. Phenol Red pH indicator in a

standard set of solutions. Far left and far right are
(b)
unacceptable and need immediate action (i.e.,
medium change, subculture or gassing.)

(c) Rotating Cell Culture System. Chamber

rotates to create simulated zero gravity so that pH6.5 pH7.0 pH7.4 pH7.6
cells do not sediment and grow as aggregates
within the chamber. (Courtesy of Synthecon.)
(d) BelloCell Culture System. Cells are grown on carriers
in center of chamber and the bellows medium chamber
(c) alternately forces medium up over beads and back down
into reservoir. (Courtesy of Bellco.)

(d)

(e)

(e) Color-coded Cryovials. Polypropylene cryostorage vials,

1.0, 1.8, 3.6, 4.6 mL. Colored inserts for caps helps to prevent
misidentification. (Courtesy of Alpha Laboratories.)

Plate 22. Medium Preparation, Culture Systems, and Cryovials.

 (a) (b) (c)
(a–c) Magnetic Sorting with Dynabeads (Dynal). Negative sort; committed progenitor cells from bone marrow suspension bound to
Dynal paramagnetic beads with antibodies to lineage markers. Lineage negative (stem) cells are not bound and remain in the
suspension ready for sorting by flow cytometry. (a) Inserting the tube into the magnetic holder. (b) Tube immediately after being placed
in magnetic holder. (c) Tube 30 s after placement in magnetic holder.

(d) (e)
(d, e) Magnetic Cell Sorting with the Midi-MACS Separator (Miltenyi Biotec). (d) Column, magnet, rack and stand. (e) Midi-MACS
separator with LD column. (Courtesy of Miltenyi Biotec.)

Plate 23. Magnetically Activated Cell Sorting.

 (a) (b)
SVpgC2a 1

SVpgC2a 2

SqCC/Y1 1

SqCC/Y1 2
Inlet/outlet to
extracapillary
NOK 1

NOK 2

GenBank space
accession no. gene (disconnected)
M92642 COL16A1
AI982638 COL16A1
Medium
M91669 COL17A1 Pump
reservoir,
X14420 COL3A1 intracapillary
M26576 COL4A1 Medium
reservoir, space
X05610 COL4A2
extracapillary
Y14690 COL5A2
space
X15880 COL6A1
M20777 COL6A2
X15882 COL6A2
L02870 COL7A1 Gas
X57527 COL8A1 exchanger
L41162 COL9A3
X17033 ITGA2
X17033 ITGA2
M59911 ITGA3
Hollow Fiber
M59911 ITGA3
Cartridge
X06256 ITGA5
S66213 ITGA6 (b) Hollow Fiber Culture. A bundle of hollow fibers of permeable plastic is
S66213 ITGA6 enclosed in a transparent plastic outer chamber and is accessible via either of
X53586 ITGA6 the two side arms for seeding cells and collecting high molecular weight
AF032108 ITGA7
product. During culture, the chamber is perfused (red arrows) from a
M14648 ITGAV
M14648 ITGAV
reservoir, via a gas exchanger and peristaltic pump, down the center of the
AF011375 ITGB4 hollow fibers through connections attached to either end of the chamber.
X53587 ITGB4 (Courtesy of FiberCell Inc; see also Fig. 26.6.)
M35198 ITGB6

(a) Affymetrix Microarray Analysis of Gene Expression. Expression of fibronectin, collagens, and integrin transcripts is detected by Affymetrix
oligonucleotide microarray in normal (NOK), SV40 T-antigenimmortalized (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocyte lines.
GenBank gene abbreviations: COL16A1 = type XVI collagen 1 chain, etc.; ITGA2 = integrin 2 subunit, etc.; ITGB4 = integrin 4 subunit, etc.
(From Sarang et al., 2003, by permission of the publisher and the authors.)

2 3
1

5
6

4
(c)
(c) The CompacT SelecT Cell Culture Robotic System. Front aspect; (1) incubator chamber with flasks on top left of carousel, (2) main
handling chamber with robotic arm in center, below left of number, capping/uncapping and dispensing unit to left of number and pipette
container below number, (3) plate incubator chamber, (4) peristaltic pumps for media additions, (5) Cedex cell counter (Innovatis), (6) multiwell
plate liquid handling unit. (Courtesy of The Automation Partnership.)

Plate 24. Automated Culture and Analysis.

 (a) (b)
(a) Mouse Embryonic Stem Cells. ES cell-like outgrowths from (b) mES Colonies Growing in 2i Serum-Free Medium. (Courtesy of Jason
cultured epiblast. (Courtesy of Jason Wray & Jennifer Nichols; Wray & Jennifer Nichols; see Protocol 23.1.)
see Protocol 23.1.)

(c) (d)
(c) Human Embryonic Stem Cells. Colony of hES cells on feeder layer (d) Human Embryonic Stem Cells. Free-floating embryoid bodies
(See also Fig. 23.2.) (Reprinted from Cooke & Minger, 2007.) (EBs). (Reprinted from Cooke & Minger, 2007.)

(e) (f)
(e) Differentiation in hES Cells. Neural differentiation after replating hES (f) Differentiation in human embryonal carcinoma cells.
cell derived neurospheres on gelatin coated plastic. Immunofluorescent Immunofluoescent staining for neural marker TUJ-1.
staining for nestin (green); nuclei stained with Hoechst 33342 (blue). (Przyborski, 2007.)
(Jackson et al., 2007.)

Plate 25. Embryo-Derived Stem Cells.

 10 mm

10 mm
(a) (b)

50 mm
(c) (d)
(a–c) MSCs from Bone Marrow Stroma. (a) Alizarin Red S-stained
monolayer showing osteogenic differentiation. (b) Oil Red O-stained (d) Multipotent stem cells from dental pulp. Adipogenic differentia-
monolayer showing adipogenic differentiation. (c) Toluidine Blue tion shown by Oil Red O staining. (Sonoyama et al., 2007.)
staining of 10 mm sections of pelleted cells. (Gregory & Prockop,
2007.) (See also Section 23.3.4.)

Plate 26. Juvenile and Adult Stem Cells.

 (a) (b)
(a) Epidermal Keratinocytes. (b) Epidermal Melanocytes.

(c) (d)
(c) Mammary Epithelium. (d) Placental Epithelium.

(e) Bronchial
Epithelium.

(f) Tracheal
Epithelium.
(e) (f)
Plate 27. Human Specialized Cells in Primary Culture (1). Cultures from first or second subcultures.
Phase contrast, 10x objective (Courtesy of Cell Applications, Inc.)
 (a)
Chondrocytes.

(b)
Osteoblasts.

(c) Internal Thoracic Smooth Muscle Myoblasts. (d) Skeletal Muscle Myoblasts.

(e) Pulmonary
Artery
Endothelium.

(f) Astrocytes.

Plate 28. Human Specialized Cells in Primary Culture (2). Cultures from first or second subcultures.
Phase contrast, 10x objective (Courtesy of Cell Applications, Inc.)