Cultivation and Retroviral Infection of
Human Epidermal Keratinocytes
There are many techniques for culturing human epidermal
keratinocytes, but the method described here
is the one devised by Rheinwald and Green (1975).
With this method, keratinocytes grow as multilayered
sheets in which proliferation is confined to the basal
layer and terminal differentiation takes place in the
suprabasal layers, thus mimicking the spatial organization
of normal interfollicular epidermis. These
cultures have a range of applications in basic research
and in the clinic. They are used to study the factors
that regulate stem cell proliferation, terminal differentiation,
and tissue assembly, as well as the events
that take place during neoplastic transformation (Jones
and Watt, 1993; Zhu and Watt, 1996; Levy et al.
Dajee et al.
, 2003). Practical applications include the
treatment of burns victims with cultured autografts
(Compton et al.
, 1989; Ronfard et al.
, 2000) and the use
of transduced keratinocytes as vehicles for gene
therapy (Gerrard et al.
, 1993; Dellambra et al.
Ortiz-Urda et al.
What follows is a description of the procedures our
laboratory uses to initiate and maintain cultures of keratinocytes
from neonatal foreskins; it is based on the
original Rheinwald and Green method, improved over
the years as described by Rheinwald (1989). The key
component of the culture system is the presence of a
feeder layer of 3T3 cells that supports the growth of
keratinocytes from clonal seeding densities (see Fig. 1).
In the laboratory, our preferred method for manipulating
gene expression in keratinocytes is by retroviral
infection (Zhu and Watt, 1996; Levy et al.
, 1998; Zhu
and Watt, 1999) and we have therefore included our
II. MATERIALS AND
|FIGURE 1 (A) Dish of cultured human keratinocytes plated
feeder layer of 3T3 J2 cells and stained with rhodanile
clones of keratinocytes are seen as large round
plaques. (B) Phasecontrast
view of clones of cultured human
keratinocytes on a feeder
layer of 3T3 J2 cells.
FAD powder [three parts Dulbecco's modified
Eagle's medium (DMEM) and one part Ham's F12
medium (F12) supplemented with 1.8 × 10-4M
adenine] is custom made by Bio Whittaker. Alternatively,
F12 (liquid, Cat. No. 41966-029) and DMEM
(liquid, Cat. No. 21765-029) can be bought separately
from Invitrogen, and adenine can be purchased from
Sigma Aldrich Co. Ltd (Cat. No. A3159). Penicillin and
streptomycin can be purchased from Invitrogen (Cat.
No.15070-063). A suitable supplier of foetal calf serum
(FCS) and bovine serum (BS) is Invitrogen. Hydrocortisone
(Cat. No. 386698) and cholera enterotoxin (Cat.
No. 227036) are purchased from Merck Biosciences
Ltd. Cholera enterotoxin is from ICN Biomedicals Ltd.
(Cat. No. 190329). Epidermal growth factor (EGF) is
purchased from Peprotech EC Ltd. (Cat. No. 100 15).
Insulin is from Sigma Aldrich Co. Ltd. (Cat. No. I5500).
Mitomycin C is also obtained from Sigma Aldrich Co.
Ltd (Cat. No. M0503). Trypsin (0.25%) (Cat. No.
M0503) and 0.02% EDTA (Cat. No. 15040-033) can be
obtained from Invitrogen. Mikrozid is purchased from
Schulke & Mayer UK Ltd. Dimethyl sulfoxide (DMSO,
Cat. No. 10323) is obtained from VWR International
Ltd. CelStirs are made by Wheaton, USA (Cat. No.
356873; Wheaton products are available through
Jencons Scientific Ltd.). Cryotubes are purchased from
Alpha Laboratories Ltd. UK (Cat. No. LW 3435).
Puromycin (Cat. No. P8833), chloroquine (Cat. No.
C6628), polybrene (Cat. No. H9268), HEPES (Cat. No.
H7006), and calcium chloride (Cat. No. C5080) are
from Sigma-Aldrich Co. Ltd. Collagen-coated dishes
are purchased from BD Biosciences. Hygromycin B (Cat. No. 10687 010) is from Invitrogen and diphtheria
toxin (Cat. No. 322326) can be purchased from Merck
A. Feeder Layer
- Culture medium for 3T3 J2 cells: This consists
of DMEM supplemented with 100 IU/ml penicillin,
100gg/ml streptomycin, and 10% bovine serum. It is
essential to batch test the serum for optimal growth of
- Trypsin-EDTA: Mix one part 0.25% trypsin and
four parts 0.02% EDTA. The same solution is used to
- Phosphate-buffered saline: To make 1 litre, dissolve
0.2g KCl, 0.2g KH2PO4, 8.0g NaCl, and 2.16g
Na2HPO4.7H2O in 900ml distilled water. Adjust pH to 7.4, add distilled water to 1-1itre final volume, autoclave,
and store at room temperature.
- Mitomycin C in PBS: Prepare a stock solution of
0.4mg/ml in PBS. Filter sterilize and store in aliquots
The J2 clone of random-bred Swiss mouse 3T3 cells
was selected to provide optimal feeder support of keratinocytes
(Rheinwald, 1989). The cells are maintained
by weekly passaging at 1:10 to 1:20 dilution. Fresh
cells are thawed every 3 months, as with prolonged
passaging the cells start to senesce or undergo spontaneous
2. Preparing the Feeder Layer
B. Keratinocyte Culture Medium
- To irreversibly inhibit proliferation, add mitomycin
C (final concentration, 4µg/ml) to confluent
flasks of 3T3 J2 and incubate for 2h at 37°C.
- Remove the medium, rinse the cells once with
0.02% EDTA, and then harvest in trypsin-EDTA. The
optimal density of the feeder layer is one-third confluent
(Rheinwald and Green, 1975; Rheinwald, 1989);
hence each mitomycin C-treated flask is split 1:3.
- The feeders can be used immediately (i.e., plated
at the same time as the keratinocytes) or prepared 1-2
days before they are required; if prepared in advance,
maintain in DMEM + 10% BS. The feeder layer should
not be prepared more than 2 days before use because
the feeder cells will start to degenerate.
- Hydrocortisone: Prepare a 5 mg/ml stock in absolute
ethanol. Store at-20°C.
- Cholera enterotoxin: Prepare a 10-5M stock in distilled
water. Store at 4°C.
- EGF: Prepare at 100gg/ml stock in FAD + FCS .
- Insulin: Prepare a 5mg/ml solution in 5mM
hydrochloric acid. Store at-20°C.
C. Source of Keratinocytes
- The basic medium consists of three parts DMEM
and one part F12 supplemented with 1.8 × 10-4M adenine (FAD), 100IU/ml penicillin, and 100µg/ml
streptomycin. Store at 4°C.
- Supplement FAD with 10% FCS. It is essential to
batch test the serum for its ability to support high
colony-forming efficiency, rapid growth, and serial
passage of keratinocytes. Serum batches optimal for
keratinocytes tend to be completely unsuitable for fibroblastic cells or hybridomas. Store FCS at-20°C before use.
- Supplement FAD + FCS further with a HICE
cocktail consisting of hydrocortisone (0.5µg/ml),
insulin (5µg/ml), cholera enterotoxin (10-10M), and
EGF (10ng/ml) (all final concentrations).
- Store complete medium (FAD + FCS + HICE) at
4°C and use within 1 week.
The usual source of keratinocytes is neonatal foreskin
obtained, with ethical approval, from routine
circumcisions. When handling any human tissue it is
essential to take appropriate precautions against the
transmission of infectious agents. Obtain the foreskin
as soon as possible after circumcision and transfer to
the laboratory dry in a sterile Bijou. If it cannot be used
immediately, store overnight at 4°C.
D. Isolation of Keratinocytes
1. PBS containing 100 IU/ml penicillin and 100µg/ml
. Other solutions are as described in
Sections III,A and III,B.
E. Passaging Keratinocytes
- Rinse the foreskin thoroughly in PBS containing
100 IU/ml penicillin and 100µg/ml streptomycin.
- Transfer the tissue to a 100-mm-diameter petri
dish, epidermis down. Remove as much connective
tissue (muscle and dermis) as possible using
sterile curved scissors.
- Transfer the epidermis and remaining connective
tissue to a fresh dish and chop into fine pieces
(1-3 mm2) using scalpels.
- Flood the dish with 10 ml trypsin-EDTA and transfer
the solution containing pieces of skin with a
wide-bore pipette to a sterile CelStir. A CelStir is
an autoclavable glass vessel containing a magnet
suspended by a rod from the lid. Solutions are
introduced and removed via a side arm in the
- Incubate the CelStir at 37°C for 30min on a magnetic
stirrer; allow the lumps of tissue to settle out
and remove the supernatant.
- Add fresh trypsin-EDTA and repeat the
- Determine the number of cells in the supernatant
with a haemocytometer. Collect the cells by centrifugation
and resuspend in FAD + FCS + HICE.
There are usually no cells from the first incubation
and then 1-5 × 106 cells per subsequent incubation.
- After four or five incubations the cell yield starts
to decline; discard any remaining lumps of tissue.
- Pool cells from each incubation and seed onto the
feeder layer at a density of 5 × 105 per 75-cm2 flask.
The average yield per foreskin is approximately
5 × 106- 107 cells.
- After 2-3 days, small colonies of keratinocytes are
visible, surrounded by 3T3 J2 feeder cells. Over
the following weeks, individual colonies expand,
displacing the feeder layer and merging with one
another. At confluence, virtually no feeder cells
remain. Feed the cells with fresh medium three
times per week.
Solutions are as described in Sections III,A and III, B.
F. Frozen Stocks of Keratinocytes and 3T3 J2
1. 10% DMSO/90% fetal calf serum
- Passage keratin0cytes prior to confluence (approximately
7-10 days after plating). Remove the medium
and rinse the cultures once with 0.02% EDTA.
- Add fresh EDTA and incubate the cultures at 37°C for about 5 min. Then selectively detach the feeders
by gentle aspiration with a pipette (Rheinwald,
- To detach the keratinocytes, add tryspin-EDTA (see
Section III,A, solution 2) to the flasks and incubate
at 37°C for about 10 min.
- Transfer the cells to a centrifuge tube; use a small
volume of culture medium to rinse the flask and
then add it to the tube, inactivating the trypsin.
- Recover the cells by centrifugation, count in a
haemocytometer, and resuspend in complete
- Cells can be passaged at a density of 1-2 × 105 per
75-cm2 flask containing 3T3 J2 feeders.
G. Preparation of Stable Packaging Lines for
- Most of the primary keratinocyte cultures are used
to prepare frozen stocks; most experiments are
carried out on second and subsequent passages.
- For freezing, harvest the keratinocytes as described
earlier, but resuspend at 106 / ml in 10% DMSO / 90%
foetal calf serum.
- Place 1 ml of cell suspension in each 1.8-ml
- Place the tubes in a rubber rack wrapped in cotton
wool overnight at-70°C and then transfer to liquid
- Freeze the 3T3 J2 cells in the same way.
- Culture medium for packaging lines: This consists of
DMEM supplemented with 100 IU/ml penicillin, 100
µg/ml streptomycin, and 10% heat-inactivated foetal
calf serum. The serum is heat inactivated in a water
bath at 56°C. for 45 min and can then be stored frozen
in aliquots. Heat inactivation of FCS improves retroviral
- Transfection solutions: 50mM chloroquine (2000x)
in dH20, 2M CaCl2 in dH20, and 2xHBS, pH 7.0. Filter
sterilise each solution, aliquot, and store at-20°C.
- 2xHBS: This is made up with 8.0g NaCl, 6.5g
HEPES, and 10ml Na2HPO4 stock solution (5.25g in
500 ml of water). Adjust the pH to 7.0 using NaOH or
HCl, bring the volume up to 500 ml, and check the pH
- Polybrene: Prepare a 5mg/ml stock solution in
PBS. Filter sterilise, aliquot, and store at-20°C.
- Puromycin: Prepare a 2mg/ml stock solution in
PBS. Filter sterilise, aliquot, and store at-20°C.
Phoenix E (ecotropic) packaging cells (Swift et al.
1999) are obtained from ATCC with prior approval
of G. Nolan (Stanford University, USA). The AM12
amphotropic packaging line was generated by
Markowitz et al.
(1988). Packaging cells are maintained
by passage at 1:5 (Phoenix E) or 1:10 (AM12) dilution.
For optimal virus production, Phoenix cells should initially
be reselected sequentially for 4 weeks, 2 weeks
in 1 µg/ml diphtheria toxin, and then two weeks in 500 µg/ml hygromycin to increase Envelope and Gag-Pol
expression (http: //www.stanford.edu/group/nolan/
index.html). Packaging cells are frozen at 106
10% DMSO/90% foetal calf serum as described (in
Section III, F,) and fresh cells are thawed every 3
months. J2 puro are 3T3 J2 cells that have been transfected
with pBabe puro to render them resistant to
puromycin (Levy et al.
Generation of Stably Transduced AM12 Cells
Packaging lines that are generated by retroviral
infection tend to have higher viral titres than those
generated by transfection (Levy et al.
, 1998). Therefore,
we use a two-step procedure in which Phoenix E cells are transiently transfected with retroviral vector, and
packaged virus released by Phoenix E cells is used to
infect AM12 cells.
H. Retroviral Infection of Cultured Human
Keratinocytes Using Viral Supernatant
- Twenty-four hours prior to transfection, plate
5 × 106 Phoenix E cells into one 10-cm-diameter petri
dish in 10 ml medium.
- Add chloroquine (final concentration 25µM) to
each plate 5 min before transfection. Add 20µg retroviral
vector DNA (we routinely use pBabe puro;
Morgenstern and Land, 1990; Levy et al., 1998) to 61 µl
2M CaCl2 and make up to a 500µl total volume with
ddH20. Add 500µl 2×HBS quickly and then bubble
vigorously with an automatic pipettor for 15s. Add
the HBS/DNA solution dropwise into the Phoenix E
culture dish and then rock the plate gently a few times
to distribute DNA/CaPO4 particles evenly. Transfer
the plate to a 37°C incubator.
- Twenty-four hours posttransfection, change the
culture medium. Gently add 6 ml of fresh medium per
10 cm plate. Leave cells overnight in a 32°C incubator;
retroviruses are more stable at 32°C than at 37°C. In
addition, seed 2 × 105 AM12 cells per 10cm plate in
10ml medium and incubate overnight at 37°C.
- The next day (i.e., 48 h posttransfection) harvest
virus by collecting the medium from Phoenix E
cultures. Gently add fresh medium to cells and
harvest virus again 8h later. Add fresh medium
for a third time, incubate the cells overnight at 32°C and collect the medium. All three supernatants
contain active virus. Each viral supernatant should
be filtered using a 0.45-µm filter on collection and
either used immediately (i.e., added to AM12 cells) or
stored at-80°C (titre will halve on each freeze/thaw
- Virus harvested from Phoenix E cells is used to
infect AM12 cells. Remove 8ml medium from the
AM12 culture. Add 5 µl polybrene (5mg/ml) and
3ml viral supernatant to each plate (5ml total
volume), shake gently, and place in a 32°C incubator
for 24 h.
- Twenty-four to 48 h postinfection the AM12 cells
are 60-80% confluent and are ready to be selected by
adding 2µg/ml puromycin to the medium. Change
medium every 2-3 days. Once the cells are confluent,
passage them, prepare frozen cell stocks, and use the
rest to infect keratinocytes.
- For some applications, AM12 are subjected to
further selection to achieve maximal viral titres. This
can be achieved by either cloning at limiting dilution
or, if there is a suitable surface marker, FACS sorting
of bulk populations. Both methods have been
described previously (Levy et al., 1998).
J2 puro are 3T3 J2 cells that have been stably transfected
with pBabe puro to render them resistant to
puromycin (Levy et al.
, 1998). They are handled in the
same way as 3T3 J2 cells, except that the medium is
supplemented with 2 µg/ml puromycin.
Retroviral Infection of Cultured Human
Keratinocytes by Coculture with Packaging
- Grow infected AM12 packaging cells to 95% confluence
and transfer to keratinocyte culture medium.
Collect virus over a 24- to 48-h period at 32°C, harvesting
virus every 8-12 h and replacing with fresh medium.
The virus should be filtered through a 0.45-gm filter on
collection and either used immediately or stored at
-80°C (titre will halve on each freeze/thaw cycle).
- Keratinocytes with the greatest proliferative
potential (putative stem cells) can be enriched by rapid
adhesion to collagen (Jones and Watt, 1993). Selection
of keratinocytes on collagen prior to infection increases
the retroviral infection efficiency to 60-80%. Harvest
subconfluent keratinocytes, first removing the 3T3 J2
feeders. Plate 2 × 106 keratinocytes per 10-cm collagencoated
plate (or equivalent plating density if smaller
dishes are used). Allow cells to adhere for 15-20min.
Wash gently with PBS and add fresh keratinocyte
medium and mitomycin C-treated J2 puro cells. The
final volume of medium should be 10 ml per 10-cm
dish. Incubate cells overnight at 37°C.
- The next day, remove 8 ml of medium and add
3ml of retrovirus supernatant and 5gl polybrene
- Incubate cells overnight at 32°C. The next day
remove virus containing medium and add fresh keratinocyte
- Infected keratinocytes are ready to use or can be
transferred to puromycin containing medium and passaged
- Prepare AM12 by treatment with mitomycin C in
the same way as for 3T3 J2 cells (Section III,A).
- Plate keratinocytes in the same way as when plating
on 3T3 J2 cells (Section III,E). Use complete keratinocyte
- After 2-3 days remove AM12, with EDTA treament,
as for 3T3 J2 cells (Section III,E). Add mitomycin
C-treated J2 puro and supplement the culture
medium with puromycin.
It is possible to obtain up to 100 population doublings
of neonatal foreskin keratinocytes prior to
senescence (Rheinwald, 1989). The number of passages
prior to senescence varies between cell strains: 5 is the
minimum, 10 is the average, and greater than 20 is the
The same basic culture procedure can be used to
grow keratinocytes from adult epidermis (although
the number of population doublings obtained will be
somewhat reduced) and from other stratified squamous
epithelia, such as the lining of the mouth and the
exocervix (Rheinwald, 1989).
Methods for transfecting Phoenix cells and retrovirally
infecting AM12 cells are based on those of Swift et al.
index.html). We routinely use the pBabe puro retroviral
vector (Morgenstern and Land, 1990). Puromycin is
the optimal drug for selection, as it is not toxic to transduced
keratinocytes and kills nontransduced cells
within 2-4 days (Levy et al.
, 1998). Following culture
and selection, the proportion of stably transduced cells
obtained with the supernatant and coculture methods
are equal (>90%). The advantage of the supernatant
method is that large numbers of cells are infected
simultaneously; however, with the coculture method,
fewer keratinocytes are required initially.
- Under the conditions described, fibroblast contamination
of keratinocytes is very rarely a problem
because the feeder layer and culture medium suppress
the growth of any human fibroblasts isolated from the
skin at the same time as the keratinocytes (Rheinwald,
1975). We have noted that some strains of keratinocytes
contain ndk-like (for nondifferentiating keratinocyte;
see Adams and Watt, 1988) epithelial cells,
but these cells are not abundant and can usually be
removed with EDTA.
- When keratinocytes are plated in culture there
is selective attachment of the basal cells, but within
1 day the cultures consist of a mixture of basal and
terminally differentiating keratinocytes (e.g., Jones
and Watt, 1993). Seeding keratinocytes at high density
appears, in our experience, to promote terminal differentiation
and is not, therefore, the answer if you are
in a hurry to obtain more cells.
- Because keratinocytes are maintained in culture
for long periods it is essential to be scrupulous in
sterile technique and laboratory cleanliness to avoid fungal or bacterial contamination. We spray our incubators
three times a week with Mikrozid.
- It is essential to be well organised and to plan
your experiments in advance. It takes about 10 days
from plating for keratinocytes to be ready for use and
sufficient feeders must be available on the days when
keratinocytes are ready for passaging.
- Use of an amphotropic retrovirus is potentially
hazardous because the virus can infect human cells.
Special care should therefore be taken and appropriate
local safety guidelines followed.
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