Human Cell Culture Protocols

538 Pages·1996·42.168 MB·English

by  Gareth E. Jones

Establishment and Maintenance
of Normal Human Keratinocyte Cultures
Claire Linge
1. Introduction
Keratmocytes are the major cellular component of the eptdermis, which is
the strattfied squamous epttheha forming the outer-most layer of skin. The
keratmocytes lie on a basement membrane and are organized mto drstmct cell
layers which differ morphologically and biochemically These regions from
the basement membrane outward are the basal, spmous, granular, and cormtied
layers. Cellular proliferation takes place mamly m the basal layer. On division,
keratmocytes give rise to either replacement progenitor cells and/or cells that
are committed to undergo the process of terminal differentiation These latter
cells leave the basal layer and gradually migrate upward, simultaneously pro-
gressing along the differentiation pathway as they go. Finally they reach the
outer surface of the epidermis m the form of fully mature functional cells, the
corneocytes. The function of these mature cells is the protectton of the under-
lying viable tissues from the external milieu.
The reasonsf or studying keratmocytesa re many-fold, and include mvesttgatton
of the pathogenesis of keratmocyte-related diseases and also examination of
the control mechanisms of proliferation and differentiation. The development
of a long-term m vitro keratmocyte system, allowing precise experimental
mampulatton of these cells, has been mstrumental in the rapid advances made
m these fields over the last two decades.
Initial attempts to grow keratmocytes were limited to the use of organ and
explant cultures (1). Using these techniques, whole pieces of skm can be kept
ahve in the short term, and growth confined to the tissue fragment or onto
IS
the plastic surroundmg the explant. However, these cultures have an extremely
short life-span and also limited apphcatron, since mixed cultures of keratmocytes
From Methods m Molecular Medune Human CeN Culture Protocols
Edlted by G E Jones Humana Press Inc , Totowa, NJ
1
2 Lmge
and fibroblasts are obtamed. Fibroblasts present a major problem where
keratmocyte culture is concerned. Even small amounts of tibroblast contami-
nation can lead to their overgrowth of keratmocyte cultures This is because of
the htgh proltferation rate of tibroblasts compared with that of keratmocytes
even under opttmum conditions for keratinocyte growth.
The greatest advance m the development of a long-term keratmocyte culture
method came m 1975, when Rhemwald and Green reported the serial culttva-
tton of pure cultures of keratmocytes from a single-cell suspension of eptder-
ma1 cells (2). Thts was achieved by growing the cells m serum-contammg
medmm on a mesenchymal feeder cell layer (trradtated mouse 3T3 cells) Usmg
this feeder layer of viable, yet nonproltferatmg, mesenchymal cells, reduced
fibroblast contammation and growth vastly, if not completely, but enhanced
the proliferation of keratmocytes The longevity of these keratmocyte cultures
was further improved on the addition of a variety of mitogens discovered to be
important for the health and growth of keratmocytes A list of these cytokmes
and the relevant references are given m Section 2. The most vttal of these
mttogens 1s eptdermal growth factor (EGF) (3)
Smce the mtroductton by Rhemwald and Green of a method of long-term
culture of keratmocytes, alternattve culture methods have been developed, each
being designed for specific experimental requirements. The degree to which
the pattern of keratmocyte dtfferenttatton m vtvo IS reproduced m vitro depends
on the conditions and methods of culture Keratmocyte cultures can vary from
undifferentiated monolayers (4) under low calcium condtttons (~0.06 mM) to
fully differentiated stratified multtlayers achieved when grown m skm-equtva-
lent cultures (5-s). Skm equivalents have been fashtoned by growmg keratmo-
cytes on the followmg
1 Collagen disks. Collagen-based, thin permeable membranes produced by ICN Flow
2 ECM gels Usually collagen-based, but can contain other extracellular matrix
(ECM) constituents, such as lamnun or fibronectm
3 Dermal equivalent ECM gels that contain viable mesenchymal cells, such as
dermal fibroblasts or fibroblastic cell lines
4 DED De-epidermized dermis Produced by multiple freeze-thawing of a piece
of skin, after which the dead epidernns can simply be peeled off This treatment
kills off all endogenous cells, leaving an uninhabited connective tissue skeleton,
which can then be reseeded with cells of the experimenters choice
The more closely the culture condtttons duplicate the ttssue environment
(i.e., actdtc pH, collagenous substrate, presence of mesenchymal cells, au
interface, etc.), the more complete the expression of eptdermal dtfferenttatton
characteristtcs.
The method detailed in this chapter is adapted from that of Rheinwald and
Green, and allows the long-term maintenance of keratmocytes in culture sup-
Human Keratwcyte Cultures 3
plymg a stock of healthy cells, which can be used either as they are or m any of
the alternative methods mentioned for experlmentatlon
2. Materials
1 3T3 cells available from the European Collection of Animal Cell Cultures
(ECACC #8803 1146)
2 3T3 medium Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal calf serum (FCS), 4 mA4 L-glutamme, 100 U/mL pemcillm,
100 pg/mL streptomycm The shelf life of this medium IS approx 4-6 wk
Owing to the Instability of L-glutamme at 4”C, however, fresh L-glutamine
can be added Media supplements are stored as concentrated stocks at -2O”C, and
media (both basic and supplemented) are stored at 4°C Note All reagents are
available from tissue-culture retailers FCS should be batch tested to obtain opt]-
mum serum for cell growth
3 Freshly isolated normal human skm m the form of abdominal or breast reduc-
tions or circumcisions
4 Skm transport medium DMEM supplemented with* 10% FCS, 100 U/mL pemcll-
lin, 100 pg/mL streptomycin, 2 5 pg/mL fungizone, 50 pg/mL gentamicin
Media supplements are stored as concentrated stocks at -2O”C, and media (both
basic and supplemented) are stored at 4°C
5 Keratmocyte growth medium (KGM) made up of a 3 1 (v/v) mixture of Ham’s
F12 and DMEM media, supplemented with 10% FCS, 4 mM L-glutamrne,
100 U/mL pemclllm, 100 pg/mL streptomycin, 0 4 pg/mL hydrocortlsone (9),
10-‘“Mcholera enterotoxm (lo), 5 pg/mL transferrm (II), 2 x lo-“M hothyromne
(II), 1 8 x 10dA4 adenme (12), 5 pg/mL msulm (1 I), and 10 ng/mL EGF (9)
This media should be used fresh If possible, but has a shelf life of approx 1 wk
Media supplements are stored as concentrated stocks at -20°C, and media (both
basic and supplemented) are stored at 4°C Stock supplements are usually made
up m phosphate-buffered salme (PBS) contammg a carrier protein, such as 0 1%
bovine serum albumin Certain media supplements require dlssolvmg as follows
Na tn-lodothyromne dissolves mltlally m 1 part of HCl and 2 parts ethanol,
ademne dissolves m NaOH, pH 9 0, msulm dissolves m 0 05M HCl, and hydro-
cortisone dissolves m EtOH
6 PBS* All PBS referred to m this text lacks calcium and magnesmm Ions and IS
made up of the followmg 1% (w/v) NaCl, 0 025% KCl, 0 144% Na2HP04, and
0 025% KH2P0, This solution IS pH-adjusted to 7 2, autoclaved at 12 1“ C ( 15
psi) for 15 mm, and stored at room temperature.
7 Trypsmizatlon solution 1 vol of trypsm stock IS added to 4 vol of EDTA stock
and used lmmedlately. Trypsm stock. trypsm (Dlfco, Detroit, MI 1 250) IS made
up of 0 25% (w/v) Trls-salme, pH 7 7 (0 8% NaCl, 0 0038% KCI, 0 01%
Na*HPO,, 0 1% dextrose, 0 3% trlzma base). Stocks are filter sterilized and stored
ahquoted at -2O’C EDTA stock EDTA 1s made up of 0 02% (w/v) EDTA m Ca-
and Mg-free PBS, autoclaved at 121°C (15 psi) for 15 min, and stored at room
temperature
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8 The followmg sterile equipment 1s reqmred: forceps, scalpel, lrls scissors, hypo-
dermlc needles, medlcal gauze, tissue-culture flasks, and Petri dishes
9 Mltomycm-C stock solution dissolve m sterile H,O to a concentration of 400
pg/mL Store at 4°C m the absence of light. Solution IS stable for 34 mo
10. Dlspase medium 3T3 medmm contammg 2 mg/mL Dlspase (Boehnnger
Mannhelm, Mannhelm, Germany) and filter sterlhzed Use unrnedlately
3. Methods
3.1. Routine Maintenance of 3T3 Cell Line
These adherent cells are grown m 3T3 media at 37°C to near confluence
(see Note 1) and passaged as follows
1 Remove media from flask, and wash cells with an equivalent volume of PBS
2 Add the trypsmlzatlon mixture to the flask at approx 1 5 mL/25 cm2 surface area,
and mcubate at 37°C for approx 5 mm, or until all cells have rounded up
3 Add 4 vol of medium to deactivate the trypsm and EDTA, and disperse the cells
with repeated ptpetmg
4 Estimate the cell number usmg a hemocytometer, and pellet the cells at approx
300g for 5 mm
5 Resuspend the cells m fresh media, and seed mto flasks or Petri dishes at approx
3 x 1O 3 cells/cm* of surface area. Note Density of cells at seeding can be varied
depending on when confluence IS required.
6 Cells should reach confluence m approx 3-5 d
3.2. Production of Feeder Layers
1 Select flasks of exponentially growmg 3T3, which have no more than 50% of
the flask’s surface area covered by cells, replace the media, and Incubate for
a further 24 h
2 Add approx l-l 0 pg of mltomycm-C/ml of medium (see Note 2), and incubate
for a further 12 h
3. Wash the flask three times with fresh medium Incubate the cells with the final
wash for approx 1O -20 mm at 37°C
4 Harvest the cells lmmedlately by trypsmlzatlon m the usual manner (detalled m
Sectlon 3 1 ) and seed m fresh flasks at approx 2 5 x lo4 cells/cm* m keratmocyte
media (1 mL media/5 cm2 of plastic surface area)
5 Incubate at 37°C for approx 12 h to allow the cells to adhere and spread before
seeding with keratmocytes
3.3. Initiation of Keratinocyte Cultures
1 Place skm sample directly from patient (see Note 3) as sterilely as possible mto a
Universal contammg a covermg volume of skm transport media at 4°C (see Note 4)
2 Before processmg, remove skm from the transport media, submerge brlefly m
alcohol three times, and shake dry m the tissue-culture hood
Human Keratinocyte Cultures 5
3. Place skm mto a shallow stertle container (a IO-cm Petri dash IS perfect for small
skm samples), and usmg fine forceps and trts scissors, tram away the hypodermis,
I e., the adipose and loose connective ttssue, unttl only the eptdermts and the
relatively dense dermis remam (see Notes 5 and 6).
4 Flatten the skin, eptdermis down, onto the surface of the Petrt dish and usrng a
sterile scalpel, cut the skm mto long 2-3-mm thin strtps
5 Place the strips mto a Umversal contammg at least a covermg amount of dtspase
medmm, and mcubate etther overnight at 4°C or for 2-4 h at 37°C
6 After the mcubatton, remove the strtps ofskm from the dtspase media, dab excess
media off on the instde of the lid of a IO-cm Petri dish, and place the relatively
media-free strips mto the Petri dish Peel the epidermis away from the dermis
wtth two sterile hypodermic needles The eptdermts IS a semiopaque thm layer,
whereas the connecttve ttssue of the dermts will have absorbed fluid and ~111
appear as a thick swollen shghtly gelatmous layer. Thts should come away eas-
tly. If secttons remain attached, then either the strtps were too thtck or further
mcubation m dispase is required (Note This should not be a problem after mcu-
batton overmght at 4°C )
7 Place the eptdern-us strtps only mto 5 mL of trypsm stock solutton, and shake
rapidly for 1 mm. Add 15 mL of DMEM/lO% FCS to inactivate the trypsm.
Remove upper eptdermal layer pteces by ptpetmg through sterile gauze mto a
sterile universal
8 Pellet the smgle-cell suspension by centrifugatton at approx 300g for 5 mm,
resuspend m keratmocyte growth medta, and count Seed at approx 2-5 x 1O 4 viable
cells/cm2 onto the preplated feeder layers
3.4. Routine Culture of Keratinocyte Strains
Regularly change the medium twice per week.
With ttme, the 3T3 feeder cells will begm to dte and detach from the flask.
Replace these with fresh feeder cells as necessary (see Note 7).
The cultures should reach confluence within 1O -14 d It IS Important to passage
the keratmocytes before they reach confluence, 1.e , when they cover approx 7&80%
of the surface area of the flask or Petrt dish (see Note 8).
To passage the keratmocytes, proceed as described for passaging of 3T3 cells m
Section 3 1 , steps 14, with the exception that keratmocytes will take longer to
trypsunze (10-I 5 mm) and wtll requtre vtgorous agitation of the flask to detach
the rounded up cells from the surface.
Once counted, seed the keratmocytes onto fresh feeder layers at a denstty of
approx 5-50 x lo3 viable cells/cm 2 The denstty seeded depends on when
confluence ts required Healthy secondary keratmocyte cultures should reach
confluence within 7-10 d (see Note 9)
4. Notes
1 The 3T3 cell line 1s an undemanding cell lme to maintam m culture, and few
problems should be encountered by a competent tissue culturtst The only thing
Lmge
to note IS that cells that have become overconfluent and begun to pile up (I e ,
form foci) should not be used either for the contmuatlon of stocks or for the
production of feeder cells, since the cells appear to transform further and can
become resistant to lrradlatlon or mltomycm-C treatment, mamtammg their pro-
liferative ability and thereby overrunnmg keratmocyte cultures
2 The exact concentration of mltomycm-C required to produce viable, yet
nonprollferatmg 3T3 cells should be titrated, since It varies with batch Alterna-
tively, feeder cells can be produced by irradiation with approx 6000 rads using a
y-u-radiator (cobalt60) This can be performed on 3T3 cells that are either attached
to the flask surface or m suspension (depending on the size of your irradiator)
The exact dose of radlatlon required to produce viable, yet nonprohferatmg cells
must be titrated
3 Generally, keratmocyte cultures from younger patients (< 16 yr old) have a greater
growth potential It 1s advlsed that cultures should not be mltlated from patients
older than 60 yr
4 Skin samples remain viable for up to 20 h when stored m skin transport media
at 4°C
5 The density of the hypodermls varies with the biopsy site For foreskins, the
hypodermls 1s particularly loose and therefore easily dissectible, whereas skm
taken from the back has an extremely dense hypodermls, which proves difficult
to remove In the latter case, Just remove as much extraneous connective tissue as
possible
6 Skm 1s often contaminated with bacteria or yeast Submerging the skm sample m
alcohol before processmg should kill most forms of contamination However,
pockets of bacteria which have become trapped m sweat or sebaceous pores may
be present Foreskins are particularly prone to blocked pores. Fortunately, once
the skm IS stretched upside down across the Petri dish, the presence of blocked
pores IS usually obvious The affected areas of skm should be carefully dissected
out and discarded, taking particular care not to cut mto the blocked pore
7 An adequate feeder layer density 1s extremely Important for the continued growth
of keratmocytes and the reduction of fibroblast growth A good feeder layer
should cover approx 70% of the surface area
8 In order to mamtam healthy cultures of rapidly growing keratmocytes (see Fig
1), It IS Imperative that keratmocyte cultures are passaged well before full
confluence 1s reached, when approx 70% of the flask’s surface area IS covered
with keratmocytes If this IS not done, then the underlying proliferative
keratmocytes will begin to die off or differentiate This is presumably owing to
the relatively impermeable multiple layers of differentiating keratmocytes reduc-
mg the nutrients available to the basal layer
9 Keratmocyte stocks can be successfully stored m liquid nitrogen Only cultures
of rapidly growing keratmocytes should be chosen for freezing, 1 e , select flasks
where only 50% of the surface area 1s covered by keratmocyte colonies
Trypsmlze, count, and pellet the cells as usual Resuspend the cells at approx
l-5 x lo6 cells/ml m 90% FCS and 10% dlmethyl sulfoxlde, place mto cryotubes
Human Keratinocyte Cultures 7
Fig. 1. A 7-d-old primary culture showing a healthy keratinocyte colony (center)
surrounded by dying feeder cells. Note the symmetrical appearance of the colony, the
smooth rounded edges of which are typical of rapidly growing keratinocyte colonies.
The phase-bright debris located at the center of the colony is commonly seen, particu-
larly in primary cultures, and is thought to be owing to cellular aggregation of termi-
nally differentiating cells to the proliferating cells before the latter adhere to the plastic
and begin to grow. Magnification 200x.
immediately, insulate tubes (wrap in multiple tissue layers or place within poly-
styrene container), and freeze overnight at -80°C before placing into liquid
nitrogen.
References
1. Cruickshank, C. N., Cooper, J. R., and Hooper, C. (1960) The cultivation of cells
from adult epidermis. J. Invest. Dermatol. 34,33!9-342.
2. Rheinwald, J. G. and Green, H. (1975) Serial cultivation of strains of human epi-
dermal keratinocytes: the formation of keratinizing colonies from single cells.
Cell 6,33 1-344.
3. Rheinwald, J. G. and Green, H. (1977) Epidermal growth factor and the multipli-
cation of cultured human epidermal keratinocytes. Nature 265,42 l-424.
4. Boyce, S. T. and Ham, R. G. (1983) Calcium regulated differentiation of normal
epidermal keratinocytes in chemically defined clonal culture and serum-free serial
culture. J. Invest. Dermatol. 81,33sAOs.
5. Bell, E., Sher, S., Hull, B., Merril, C., Rosen, S., Chamson, A., Asselineau, D.,
Dubertret, L., Coulomb, B., Lapiere, C., Nusgens, B., and Neveux, Y. (1983) The
reconstitution of living skin. J. Invest. Derm. 81,2s-10s.
8 Linge
6 Prunetras, M , Regmer, M , and Woodley, D (1983) Methods of cultivation of
keratmocytes at an au liquid mterface J Invest Dermatol 81, 28s-33s
7 Boyce, S T , Chrtsttanson, D J., and Hansborough, J F (1988) Structure of a
collagen-glycosammoglycan dermal skm substitute opttmtsed for cultured human
eptdermal keratmocytes J Boomed Mater Res 22, 939-957
8 Yannas, I V , Lee, E , Orgtll, D P , Skrabut, E M , and Murphy, G F (1989)
Synthesesa nd charactertsatton of a model extracellular matrix that induces partial
regeneration of adult mammahan skm Proc Nat1 Acad SCI USA 86, 933-937
9 Rhemwald, J G (1980) Serial cultrvatton of normal human eptdermal keratmo-
cytes Methods Cell Brol 21, 229-254
10 Green, H (1978) Cychc AMP m relation to prohferatton of the eptdermal cell a
new vtew Cell 15,801-811
11 Watt, F M and Green, H (198 1) Involucrm synthesis IS correlated with cell size
m human eptdermal cultures J Cell Blol 90, 738-742
12 Wu, Y J., Parker, L. M , Binder, N E , Beckett, M A , Smard, J H , Grtffiths, C
T , and Rhemwald, J G (1982) The mesotheltal keratms a new family of
cytoskeletal protems identified m cultured mesothehal cells and non-keratmtsmg
epttheha Cell 31, 693-703
Cultivation of Normal Human Epidermal Melanocytes
Mei-Yu Hsu and Meenhard Herlyn
1. Introduction
An Important approach m studies of normal, diseased, and malignant cells IS
their growth m culture. The lsolatlon and subsequent culture of human eplder-
ma1 melanocytes has been attempted since 1957 (l-5), but only since 1982
have pure normal human melanocyte cultures been reproducibly established to
yield cells m sufficient quantity for bIological, biochemical, and molecular
analyses (6). Selective growth of melanocytes, which comprise only 3-7% of
epldermal cells in normal human skin, was achieved by suppressing the growth
of keratmocytes and fibroblasts m epldermal cell suspensions with the tumor
promoter 12-O-tetradecanoyl phorbol- 13-acetate (TPA) and the Intracellular
cyclic adenosme 3’, 5’ monophosphate (CAMP) enhancer cholera toxin, respec-
tively, which both also act as melanocyte growth promoters. Recent progress
in basic cell-culture technology, along with an improved understanding of cul-
ture requirements, has led to an effective and standardized isolation method,
and special culture media for selective growth and long-term maintenance of
human melanocytes. The detailed description of this method IS aimed at
encouragmg its use in basic and applied blologlcal research
2. Materials
1 Normal skm-transportmg medmm The medmm for collectmg normal skm IS
composed of Hank’s balanced salt solution (HBSS without Ca2+ and Mg2+,
Glbco-BRL [Grand Island, NY], #2 1250-089) supplemented with pemclllm (100
U/mL, USB [Cleveland, OH], #199B5), streptomycin (100 pg/mL, USB,
#2 1 B65), gentamlcm (100 pg/mL, BloWlttaker [Walkersvllle, MD], #17-5 1S Z),
and funglzone (0 25 pg/mL; JRH Biosciences [Lenexa, KS], #59-604-076) After
sterlhzation through a 0 2-pm filter, the skm-transporting medmm IS transferred
mto sterile containers m 20-mL ahquots and stored at 4°C for up to 1 mo
From Methods In Molecular Medune Human Cell Culture Prorocols
Edlted by G E Jones Humana Press Inc , Totowa, NJ
9
‘0 Hsu and Herlyn
2 Eptdermal tsolatton solutton Dilute 0 5 mL of 2 5% trypsm solutton (BIO-
Wtttaker, #17-160E) with 4 5 mL of HBSS without Ca2+ and Mg2+ at pH 7 4 to
yield a final trypsm concentration of 0 25%
3 Cell-dispersal solutton 1 25 U/mL dtspase (neutral protease, grade 11, Boehrmger
Mannhetm [Indtanapohs, IN], #295-825) 0 1% (w/v) hyaluronldase (type 1S from
bovme testis, Sigma [St Louts, MO], #H3506), and 10% heat-inactivated fetal calf
serum (FCS, Stgma, #F2442) m MCDB 153 medium (Sigma, #M7403) supple-
mented with 2 mM CaCl, and mixed wtth Letbovttz’s L- 15 at a 4 1 (v/v) ratto
4 Bovine pmutary extract (7,8) The followmg should be prepared before extraction
a Weigh out bovine pttmtary glands (Pel-Freeze Btologtcals [Rogers, AR],
#57 133-2) mto 25-30-g batches Place m Z~ploc@ bags, and store at -70°C
b Thirty liters of cold (4°C) 1X phosphate-buffered salme (PBS) wtthout Ca2+
and Mg2’
c One hter of cold 0 15MNaCl saline solution
d One ltter of 0 2 mM EDTA solution
e Prechill high-speed centrifuge rotor at 4°C
f Boll 6000-8000 Dalton dialysis tubing (10 strips about 2 ft in length,
Spectrum Medical Industries [Los Angeles, CA], #132655) twtce In
ddH,O and once m EDTA solution (prepared m Section 2 . step 4d) Boil
for 20 mm each time Leave tubing m beaker filled with 1X PBS, and store
at 4°C for up to 3 d
The bovine pmntary extract IS prepared as follows (steps g-p are performed m a
cold room)
Thaw and rinse pttuttary glands m ddH20, handling each batch of prewetghed
g
pituitary glands separately
h Pulse-blend thawed pttmtary glands in a blender containing 2 38 mL cold
salme solutton/g of pmutary gland to break up large pieces Pulses should not
exceed 30 s because the temperature must remam low Transfer the resulting
mixture mto a 2-L flask
1 Repeat steps g and h for all batches of pttmtary glands
J Stir the pooled mixture m the 2-L flask for 90 mm
k Pour the mixture mto plastic centrifuge bottles
I Spm at 12,000g for 45 mm at 4°C m a prechtlled rotor to remove debris
m Plpet supernatant from bottles into dialysis tubmg, and begm dtalysts agamst
cold 1X PBS m a cold room
n Change the buffer three times m 3 d
0 Filter sequentially through low protem-bmdmg filters of 0 45 urn (Mtlhpore
[Marlborough, MA], SLHV025 LS) to eliminate any fragments and 0 2 pm
(Milhpore, SLGV025 LS) to sterilize the filtrate Prepare 5-mL ahquots, and
store at -70°C for up to 6 mo Once thawed, pttmtary extract should be diluted
m medium tmmedtately
P Determine the protein concentratton of the extract using a protem assay ktt
(Pierce [Rockford, IL], BCA Protein Assay Reagent, #23225H), and titrate
the optimal concentratton m medium (approx 40 pg/mL)