Developments in xenobiotic-free culture of human keratinocytes for clinical use

Developments in xenobiotic-free culture of humankeratinocytes for clinical use

TAO SUN, PhD a; MIKE HIGHAM, MS a; CHRIS LAYTON, MD b; JOHN HAYCOCK, PhD a; ROBERT SHORT, PhD a;SHEILA MACNEIL, PhD a,b

We have recently reported that irradiated human fibroblasts can be used as a feeder layer, to expandkeratinocytes under serum-free conditions, on a chemically defined plasma polymer surface developed forthe culture and transfer of keratinocytes for clinical use. While this is a significant advance in developing aserum-free keratinocyte culture approach, the need to irradiate fibroblasts to growth-arrest them and preventthem from overgrowing the keratinocytes introduces another small, but potential, risk for the patient. The aimof the present study was to develop conditions for the coculture of normal human keratinocytes with non-irradiated normal human fibroblasts under serum-free conditions. We examined the fibroblast/keratinocyterelationship on three separate surfaces: tissue culture plastic, non-tissue culture plastic, and a plasma polymersurface designed for clinical use. We report that it is possible to achieve rapid and successful expansion ofhuman keratinocytes under serum-free conditions on all three surfaces providing one uses a keratinocyte-friendlymedia, a minimum seeding density of keratinocytes, and a ratio of fibroblasts to keratinocytes that does notexceed 1 : 1. These results provide us with a rapid laboratory expansion of proliferative human keratinocytes,under completely defined culture conditions, without any xenobiotic cells (mouse fibroblasts) or material (bovineserum), for the treatment of patients with extensive skin loss. (WOUND REP REG 2004;12:626–634)

Rapid laboratory expansion of proliferative humankeratinocytes can be a valuable aid in the treatment ofpatients with extensive skin loss. The current methodof culturing keratinocytes for clinical use, in use sincethe 1980s, employs gamma-irradiated mouse 3T3 fibro-blasts (growth-arrested) as a feeder layer in media(Green’s media) containing 10% bovine fetal calfserum.1 However, with growing concerns about the

transmission of bovine spongiform encephalitis (BSE)and some murine viruses, it would be preferable toculture cells under completely defined culture condi-tions, without any xenobiotic cells (mouse fibroblasts)or material (bovine serum).

Although bovine serum-free media have beendeveloped for keratinocyte proliferation, some ofthese media use bovine pituitary extracts as a bovineserum substitute.2 This would not be viewed as redu-cing the risk of transmission of BSE in European coun-tries where BSE has been detected.3 The bovine serumsupplies mitogens for the cells, as does the irradiatedmouse fibroblast feeder layer, which also suppliesattachment factors for the keratinocytes. Other proprie-tary media that claim to be free of xenobiotic materialnevertheless do not disclose media constituents. At

BSE Bovine spongiform encephalitis

DMEM Dulbecco’s modified Eagle medium

FCS Fetal calf serum

PBS Phosphate buffered saline solution

From the Department of Engineering Materialsa, SheffieldUniversity and Division of Clinical Sciencesb,Sheffield University, Northern General HospitalSheffield, United Kingdom.

Manuscript received: December 17, 2004Accepted in final form: July 15, 2004Reprint requests: Sheila MacNeil, Professor of Cell and

Tissue Engineering, Department of EngineeringMaterials, Sir Robert Hadfield Building, MappinStreet, Sheffield, S1 3JD, United Kingdom. Fax:þ44 114 222 5943; Email: [email protected].

Copyright # 2004 by the Wound Healing Society.ISSN: 1067-1927 $15.00 + 0.

626

present none of these media are ‘‘approved’’ for clinicaluse to the best of our knowledge.

In the presence of a fibroblast feeder layer,keratinocytes expand rapidly and maintain their col-ony-forming potential. In the absence of fibroblasts,keratinocytes quickly begin to undergo terminaldifferentiation.4 Similarly, in three-dimensional recon-structed human skin, fibroblasts also play a crucialrole. In the absence of fibroblasts, keratinocytes arepoorly attached to the dermis and disorganized. Withfibroblasts present, there is good attachment, remodel-ing of the basement membrane, and keratinocytes takeon epithelial organization.5

We have recently reported that irradiated humandermal fibroblasts can be used as a feeder layer toexpand keratinocytes under serum-free conditions6 ona chemically defined plasma polymer surface devel-oped for the culture and transfer of keratinocytes forclinical use.7–10 While this is a significant advance indeveloping a serum-free keratinocyte culture approach,the need to irradiate fibroblasts to growth-arrest themand prevent them from overgrowing the keratinocytesintroduces another small but potential risk for thepatient.

Accordingly, the aim of the present study was toextend this work to develop conditions for the cocul-ture of normal human keratinocytes with nonirradiatednormal human fibroblasts under serum-free conditions.We examined the fibroblast/keratinocyte relationshipon three separate surfaces: tissue culture plastic,non-tissue culture plastic, and a plasma polymersurface designed for clinical use in the absence ofserum. We report that it is possible to achieve rapidand successful expansion of human keratinocytesunder serum-free conditions on all three surfaces ifone uses media that supports keratinocyte culturewith a minimum seeding density of keratinocytes anda ratio of nonirradiated fibroblasts to keratinocytes thatdoes not exceed 1 : 1.

MATERIALS AND METHODSNormal human keratinocytes and fibroblasts wereisolated from skin obtained from routine operations(abdominoplasty and breast reduction) according tolocal Ethical Committee guidelines (National HealthService Trust, Sheffield, UK). For standard culture,keratinocytes were freshly isolated from the dermal/epidermal junction after trypsinization (4 ºC, 18 hours;DIFCO Laboratories, Detroit, MI) following the methodof Goberdhan et al.,11 which uses Green’s media plus10% fetal calf serum (FCS). All fibroblasts wereinitially established as described previously inDulbecco’s modified Eagle medium (DMEM; ICN Flow,Thame, UK) containing 10% FCS (Advanced ProteinProducts, Brierley Hill, UK).12 Briefly, the dermal

remains after trypsinization for keratinocyte extractionwere washed in phosphate buffered saline solution(PBS; Oxoid, Unipath, UK), finely minced, and incubatedin 0.5% (w/v) collagenase A (Boehringer-Mannheim,Lewes, UK) at 37 ºC for 16 hours. Fibroblasts obtainedfrom this enzyme digest were expanded in DMEMsupplemented with 10% (v/v) fetal calf serum, 2 · 10�3

mol/L glutamine, 0.625 mg/ml amphotericin B, 100 ILU/mlpenicillin, and 100 mg/ml streptomycin (all from Gibco/Invitrogen, Paisley, UK) andwere used between passages4 and 9 for experimentation.

Keratinocytes (freshly isolated) were coculturedwith fibroblasts (initially isolated and expanded inDMEM plus FCS) in serum-free or serum-containingGreen’s medium (DMEM and Ham’s F12 medium[Gibco/Invitrogen] in a 3 : 1 [v/v] ratio supplementedwith 10 ng/ml epidermal growth factor, 0.4 mg/mlhydrocortisone, 10�10 mol/L cholera toxin, 1.8 · 10�4

mol/L adenine, 5 mg/ml insulin, 5 mg/ml transferrin,2 · 10�3 mol/L glutamine, 2 · 10�7 mol/L triiodothyr-ionine, 0.625 mg/ml amphotericin B, 100 Iu/mlpenicillin, and 100 mg/ml streptomycin [all fromSigma-Aldrich, Poole, UK, unless specified differentlyabove]).

Immunofluorescent labeling of involucrinAn adaptation of the method of Hudson et al.13 wasused to label involucrin expression in keratinocytes,which had been grown in 24-well plates. Cells werefixed with 2 % paraformaldehyde (BDH Chemicals,Poole, UK) for 10 minutes, washed with PBS (· 3) andpermeablized using 0.1% Triton X100 (BDH Chemicals)for 20 minutes. The plates were then washed with PBS(· 3) and neutralized with 50 mM ammonium chloride(BDH Chemicals) in PBS for 10 minutes. After a thor-ough wash in PBS (· 3), the cells were incubatedwith 10% (v/v) normal goat serum (Sigma-Aldrich)diluted in PBS at room temperature for 45 minutes,and then incubated with 18 mg/ml mouse IgG1 anti-human involucrin (Sigma-Aldrich) in PBS for 45minutes at room temperature. After a thorough washwith PBS (· 3), the cells were incubated with goatanti-mouse IgG-FITC conjugate (Sigma-Aldrich) inPBS (1 : 50) and 4´,6-diamidine-2-phenylindole (DAPI;Vector Laboratories, Burlingame, CA; 1 : 1000 v/v inPBS) for 45 minutes. After further washing with PBS(· 3), the fluorescent staining was visualized using theAXON image express system (Axon Instruments/Mole-cular Devices, Union City, CA). Briefly, fluorescencemicrographs of immunolabeled samples were takenusing epifluorescent illumination at lex ¼ 495 nm,lem ¼ 515 nm (for FITC/involucrin visualization) andlex ¼ 358 nm, lem ¼ 461 nm (for DAPI/nuclei visuali-zation). Negative control samples were processed bysubstituting the primary antibody with preimmune iso-type IgG antibody.

WOUND REPAIR AND REGENERATIONVOL. 12, NO. 6 MACNEIL 627

Immunolabeling of pan-cytokeratinCells were cultured on coverslips and fixed with 2%paraformaldehyde for 10 minutes, washed three timeswith 1· wash buffer (0.05 M Tris containing 0.3M NaCland 0.1 % Tween 20, 10·; Dako, Carpintera, CA). Thecells were then incubated with blocking buffer (0.05 MTris-HCl buffer, pH 7.2–7.6, and 1% bovine serum albu-min [Sigma-Aldrich]) at room temperature for 45 min-utes. The blocking buffer was removed and anti-humancytokeratin, AE1/AE3 monoclonal antibody (M3515,Dako; 1 : 5–20 v/v in 0.05 M Tris-HCl buffer, pH 7.2–7.6) was added to cover the specimens for 15 minutesand then washed thoroughly. The cells were incubatedwith goat anti-mouse IgG-AP conjuate (1 : 50 v/v in 0.05M Tris-HCl buffer, pH 7.2–7.6; Dako) for 15 minutes.After a thorough wash, the substrate-chromogensolution was added to cover the specimens and incu-bated for 5–30 minutes, and then rinsed gently withdistilled water. Aqueous hematoxylin (Dako) wasadded for 20 seconds and then washed gently withdistilled water. Samples were incubated in 37 mMammonia water for 2 minutes and washed thoroughlywith distilled or deionized water. Specimens were thenmounted with Faramount (Dako) for normal lightmicrocopy analysis.

Plasma copolymerizationAcrylic acid (> 99%) and octa-1,7-diene (> 99%) mono-mers (Aldrich Chemical) were used as received follow-ing several freeze/thaw cycles. Polymerization wascarried out in a cylindrical reactor, evacuated by avacuum pump. The plasma was sustained by a radiofrequency signal generator (13.56 MHz) and amplifierinductively coupled via an impedance matching unitand an externally wound copper coil. The plasmapower was 10 W at a total flow rate of 2.0 cm3

(stp)

min�1. The reactor pressure was typically 3 · 10�2

mbar during polymerization. Plasma polymers weredeposited onto clean aluminum foil for XPS analysisand onto tissue culture well plates for cell culture. Adeposition time of 20 minutes was employed and themonomer mixtures allowed to flow for 15 minutes afterthe plasma had been extinguished. This minimizeduptake of atmospheric oxygen by the coatings uponexposure to the atmosphere.7

Assessment of colony formation by image analysisA computer software image analysis method wasemployed (Openlab v.3.0.2, Improvision, Coventry,UK) to evaluate the areas of keratinocyte and/or fibro-blast colonies. Briefly, normal light micrographs ofsamples were taken using a Leica DM-IRB invertedfluorescent microscope and colony areas calculatedusing Openlab v3.0.2.

Statistical analysisMann–Whitney nonparametric analysis of the growth ofkeratinocytes with fibroblasts on three different sur-faces was undertaken.

RESULTSThe appearance of cells in nonirradiated fibroblast andkeratinocyte cocultures is illustrated in Figure 1 (phasecontrast photomicrograph). Keratinocytes were thenspecifically identified by staining for cytokeratins (redend point) or involucrin with all cell nuclei identified bystaining for hematoxylin (blue; Figure 1-II). The nucleiof cocultured fibroblasts and keratinocytes werestained with DAPI (Figure 1-III), while Figure 1-IVshows keratinocytes stained for involucrin (green). Ascontrol experiments, separate cultures of keratinocytesand nonirradiated fibroblasts were stained with DAPIand anti-involucrin antibody, respectively (Figures 1-V,VI, VII, and VIII).

Influence of media on keratinocyte proliferationwhen cocultured with fibroblastsNonirradiated normal human fibroblasts were preparedas a feeder layer in 24-well tissue culture plates andincreasing numbers of keratinocytes (5.0 · 105, 2.5 ·105, 1.2 · 105, 6.0 · 104, 3.0 · 104 per well) were seededon top of these cells and the cultures continued for 12days. Cultures of cells in standard DMEM and Green’smedia (both supplemented with 10% FCS) was com-pared. As Figure 1-I shows, keratinocytes formed sepa-rate colonies in the presence of normal fibroblastfeeder layers in both media. The experimental data(Figures 2A and B) indicated that in both media if ahigh number of keratinocytes were seeded, keratino-cyte colonies formed rapidly. However keratinocytescontinued to do well only in Green’s media, not inDMEM media (both containing serum). As a controlexperiment, different numbers of fibroblasts wereseeded on top of a keratinocyte feeder layer, butno fibroblast colonies were detected in eithermedia within the culture period (1–7 days; results notshown).

Nonirradiated normal human fibroblasts and ker-atinocytes were then deliberately mixed in differentratios and coseeded in 24-well tissue culture plates ineither DMEM or Green’s media containing serum. Thetotal cell number in each well was fixed as 5.0 · 105.The ratios of keratinocytes to nonirradiated fibroblastswere: 10 : 0, 8 : 2, 6 : 4, and 4 : 6. Clearly separatedcolonies of keratinocytes and fibroblasts in the samewell were detected after culture for 2–3 days. Successfulkeratinocyte culture depended on both the cell ratioand the media as shown in Figures 2C and D.Under the same experimental conditions, a high ratio

WOUND REPAIR AND REGENERATION628 MACNEIL NOVEMBER–DECEMBER 2004

of keratinocytes to fibroblasts resulted in a high %age ofthe cells being keratinocytes and vice versa. DMEM-containing serum favored fibroblast growth whileGreen’s media supplemented with 10% FCS favoredkeratinocyte growth, which confirmed the previousexperimental results.

Serum-free coculture of keratinocytes andnonirradiated fibroblasts on tissue culture plasticDifferent numbers of keratinocytes (2 · 105, 8 · 104,4 · 104, 2 · 104, 4 · 103 per well) were seeded onto anonirradiated normal human fibroblast feeder layer in24-well tissue culture plates and cultured for 8 days inserum-free DMEM and serum-free Green’s media. Cul-

tures of keratinocytes (2 · 105 cells/well) on a normaltissue culture plastic surface in serum-free DMEM andserum-free Green’s media were used as control experi-ments. The experimental data (Figure 3A and B) indi-cated that if keratinocytes at > 4 · 104 cells per wellwere employed, keratinocyte colonies could occupymore than 90% of the well surface in serum-freeGreen’s media. However, in serum-free DMEM media,few keratinocyte colonies were detected within theculture period, even when the highest number of kera-tinocytes was used. In control experiments withoutfibroblasts, keratinocyte colonies rarely achievedmore than 40% of the surface area in serum-freeGreen’s media and no keratinocytes were found tosurvive in serum-free DMEM.

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FIGURE 1. Microscopic characterization ofcocu l t u r e s and pu re cu l t u r e s o fnonirradiated fibroblasts and keratinocytes.(I) Phase contrast photomicrograph ofke ra t i nocy te s and non i r r ad ia tedfibroblasts in coculture and (II) followingpan-cytokerat in immunostain ing ofkeratinocytes. (III) Keratinocytes andnonirradiated normal human fibroblastcocultures stained with DAPI and (IV)involucrin immunolabeling. (V) Pure culturesof keratinocytes stained with DAPI and (VI)involucrin immunolabeling. (VII) Pure culturesof nonirradiated normal human fibroblastsstained with DAPI and (VIII) involucrinimmunolabeling. Bar ¼100 mm.


Different numbers of nonirradiated normal humanfibroblasts (103, 5 · 103, 104, 5 · 104, and 105) and kera-tinocytes (103, 5 · 103, 104, 5 · 104, 105, and 5 · 105) werethen mixed and coseeded in 24-well tissue cultureplates in serum-free Green’s media. As Figure 4Bshows, if 1 · 105 of nonirradiated fibroblasts andmore than 1 · 105 of keratinocytes were seeded in eachwell of a 24-well tissue plate, within 7 days more than70% of the surface was occupied by keratinocytes.

Table 1 summarizes the ratio of nonirradiated fibro-blasts to keratinocyte numbers on tissue culture plasticunder serum-free culture conditions in Green’s mediawithout serum. It shows that one needs a minimumseeding density of keratinocytes and a minimum fibro-blast presence for successful keratinocyte expansion.

Too high a ratio of fibroblasts to keratinocytes limitskeratinocyte growth.

Serum-free coculture of keratinocytes andnonirradiated fibroblasts on a plasma surfaceIn this experiment, a plasma polymer containing sur-face 9.2 % COOH/R groups was used to coat 24-wellplates. Different numbers of nonirradiated fibroblasts(1.0 · 104, 5.0 · 104, and 1.0 · 105) and keratinocytes(5.0 · 104, 1.0 · 105, and 5.0 · 105) selected from earlierexperiments on tissue culture plastic (as summarizedin Table 1) were mixed and coseeded in each well inserum-free Green’s media. As Figure 5A illustrates, if5.0 · 105 of keratinocytes and 5.0 · 104 of fibroblasts

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FIGURE2. Time course of colony formation of human keratinocytes grown on, and mixed with, nonirradiated normal humanfibroblasts in DMEM and Green’s medium. (A & B) Keratinocytes were grown on a monolayer of nonirradiated human fibroblasts;(C & D) the keratinocytes were mixed with the fibroblasts prior to plating. (A) The keratinocytes cultured on fibroblast monolayerswere grown in DMEMmedium; while in (B) Green’s medium was used. The keratinocyte numbers in each well of A and B: l 5.0 · 105,* 2.5 · 105, , 1.2 · 105, . 6.0 · 104, & 3.0 · 104. (C) Mixed cultures were coseeded in 24-well tissue culture plate in DMEM medium.The total cell number in each well was 5.0 · 105. The keratinocyte and fibroblast ratios in C and D were: l 10:0, * 8:2, , 6:4, . 4:6. (D)Per (C) except cultures grown in Green’s medium. Results shown are means � standard deviation of triplicates.


were seeded in each well, within 4 days more than90% of the well surface was occupied by keratinocytes.If only 1.0 · 104 of fibroblasts were employed,the keratinocytes took 9 days to occupy 90% of thesurface. As Figure 5B shows, if 1.0 · 105 of keratino-cytes and 5.0 · 104 of fibroblasts were seeded ineach well, after 10 days of culture more than 90% ofthe well surface was occupied by keratinocytes. If only1.0 · 104 of fibroblasts were used, the keratinocytesdid not achieve a significant surface area. Theseresults essentially confirm the findings for cocultureof cells on tissue culture plastic as summarized inTable 1.

Serum-free coculture of keratinocytes andnonirradiated fibroblasts on a non-tissue culturesurfaceNonirradiated normal human fibroblasts and kerati-nocytes at 1.0 · 105 were cultured separately andtogether in non-tissue culture 24-well plates in serum-free Green’s media. As Figure 6 shows, when cells werecultured alone, the fibroblasts occupied � 50% of thesurface, and the keratinocytes less than 5% of the sur-face. However, when cultured with nonirradiated fibro-blasts, keratinocytes proliferated to cover more than95% of the non-tissue culture surface in serum-freeGreen’s media by 8 days. Again, these results are essen-tially similar to those for coculture of cells on eithertissue culture plastic (Figures 3 and 4) or the plasmapolymer surface (Figure 5). There was no statisticaldifference between keratinocyte expansion on thesedifferent substrates when comparable starting numbersof cells were assessed by Mann–Whitney nonpara-metric comparison.

DISCUSSIONThe purpose of this study was to develop a normalhuman keratinocyte and nonirradiated normal humanfibroblast coculture system under serum-free condi-tions, which could be used clinically. Of the method-ologies currently available, expansion of keratinocyteson a lethally irradiated feeder layer of mouse fibro-blasts followed by detachment of these cells as anintegrated sheet of cells using dispase is consideredto be the ‘‘gold standard’’ for laboratory expansion ofcells as an augmentation to conventional split-thickness skin grafts.1 However, with growing concernsabout the transmission of BSE and some murineviruses, it would be preferable to culture cells undercompletely defined culture conditions, without anyxenobiotic cells or materials. Recently, an irradiatedhuman fibroblast feeder layer was successfullyemployed to aid the expansion of keratinocytes in aserum-free culture system in our laboratory.6 Thisstudy is an extension of that research, in whichnonirradiated normal human fibroblasts are used as afeeder layer for keratinocyte expansion in serum-freeGreen’s media. To analyze the performance of the twocells in coculture, keratinocytes were identified byinvolucrin and pan-cytokeratin immunostaining andimage analysis software was used to calculate thearea of keratinocyte cultures achieved.

Keratinocytes can be successfully cultured on asurface coated with type I collagen and have beenused clinically; however, sources of type I collagenfor clinical use are usually bovine.14 While bovine col-lagen has US Food and Drug Administration approvalfor clinical use, European regulatory authorities wouldprefer that cells cultured for clinical use avoid the use

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FIGURE3. Growth of human keratinocytes in serum-free medium. (A) Time course of colony formation of human keratinocytes on anonirradiated normal human fibroblast feeder layer in serum free Green’s medium. The keratinocyte numbers in each well were:l no keratinocyte, * 4.0 · 103, . 2.0 · 104, , 4.0 · 104, & 8.0 · 104, & 2.0 · 105. (B) Same as A, except serum-free DMEM medium wasused. Results shown are means �standard deviation of triplicates.


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FIGURE4. Growth of human keratinocytes inmixed cultures of keratinocytes andnonirradiated human fibroblasts grown inserum-free Green’s medium. (A) Colonyformation of keratinocytes 4 and (B) 7 daysafter mixing with nonirradiated humanfibroblasts and coseeded in 24-well tissueculture plate in serum-free Green’s medium.Results shown are means of triplicates.Standard errors of the mean were of theorder of 5–10% for these experiments but arenot shown to keep the presentation visuallysimple.

Table 1. Time taken for keratinocytes to achieve 90% surface area after mixing with human fibroblasts and coseeding in coated24-well tissue culture plate in serum-free Green’s medium

Keratinocyte density Fibroblast density

(cells/well) (cells/well) Time to achieve keratinocyte density of 90 % (d)

5.0 · 105 1.0 · 105 3–45.0 · 105 5.0 · 104 3–55.0 · 105 1.0 · 104 9

1.0 · 105 1.0 · 105 71.0 · 105 5.0 · 104 81.0 · 105 1.0 · 104 Not achieved

5.0 · 104 1.0 · 105 Not achieved5.0 · 104 5.0 · 104 Not achieved5.0 · 104 1.0 · 104 Not achieved

Results shown are means of triplicates.


of bovine- and other animal-derived products, to reducethe risk of disease transmission. The main finding ofthis study is that it is possible to get rapid expansion ofkeratinocytes on tissue culture plastic or on non-tissueculture polystyrene surface or on a plasma polymersurface containing 9.2 % COOH/R groups underserum-free conditions if one has a minimum seedingdensity of keratinocytes, an appropriate ratio of normalnonirradiated human fibroblasts, and keratinocyte-friendly media.

When nonirradiated normal human fibroblasts andkeratinocytes were cocultured in Greens’ media andDMEM media with 10% serum, the two cells werefound to form separate colonies, even when initiallymixed together prior to seeding. If the keratinocyteratio was high enough, more than 90% of the well surfacewas occupied by keratinocytes in both media. In serum-free conditions, however, rapid expansion of keratino-cytes in the presence of fibroblasts was only found inGreen’s media. It appears that the serum-free Green’smedia and normal nonirradiated human fibroblasts pro-vide all the necessary growth factors and extracellularmatrix proteins needed to support keratino-cyte growth.This data shows that one of the old maxims of culture ofkeratinocytes—that fibroblasts, unless growth restricted,will always overgrow keratinocytes—is not always true.This article illustrates that one can readily obtain kerati-nocyte expansion, serum free, with nonirradiated fibro-blasts, and we show that this can be achieved on threedifferent substrates. Three substrates were compared inthis study to establish whether the epithelial/mesenchy-mal biology was generic, and this indeed appears to bethe case.

The potential of serum-free coculture of normalhuman keratinocytes and nonirradiated normal humanfibroblasts on plasma polymers is exciting. It providesan opportunity to develop a xenobiotic-free culture andcell delivery system. The next step will be to achieveinitial serum-free expansion of human fibroblasts andthen to examine the ability of keratinocytes to transferfrom fibroblast/keratinocyte cocultures to an in vitrowound bed model,8 work that is ongoing in our labora-tory at present.

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FIGURE5. Effect of fibroblast number on the growth of humankeratinocyte colonies in mixed cultures of keratinocytes andhuman nonirradiated fibroblasts. (A) Time course of colonyformation of human keratinocytes after mixing withnonirradiated human fibroblasts and coseeding in coated24-well tissue culture plate in serum free Green’s medium. Thekeratinocyte number in each well was fixed at 5.0 · 105. Thefibroblast numbers were 1.0 · 105, 5.0 · 104, 1.0 · 104. (B) Sameas A except the keratinocyte number in each well was fixedas 1.0 · 105. Results shown are means of triplicates. Standarderrors of the mean were of the order of 5–10% for theseexperiments but are not shown to keep the presentationvisually simple.

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FIGURE6. Colony formation of nonirradiated human fibroblastculture, keratinocyte culture, and nonirradiated fibroblast/keratinocyte coculture in a 24-well non-tissue culture plate inserum-free Green’s medium. The keratinocyte number in eachwell was fixed as 1.0 · 105. The fibroblast number in each wellwas fixed as 1.0 · 105. Results shown are means of triplicates.Standard errors of the mean were on the order of 5–10% forthese experiments but are not shown to keep the presentationvisually simple.


In summary, the current study shows that the add-ition of nonirradiated human fibroblasts to the cultureof human epidermal keratinocytes on tissue cultureplastic or a 10 W plasma polymer surface containing9.2 % COOH/R groups allows keratinocyte proliferationunder serum-free conditions.

ACKNOWLEDGMENTSWe acknowledge financial support from the EPSRC(UK) for Dr. Tao Sun for this study and of an EPSRC-funded studentship for the support of Dr. Mike Higham.

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Documents

Developments in xenobiotic-free culture of human keratinocytes for clinical use