Journal of Surgical Research 152, 296–302 (2009)

Wound Healing on Athymic Mice With Engineered Skin SubstitutesFabricated with Keratinocytes Harvested from an Automated Bioreactor

Balaji Kalyanaraman, M.B.B.S.,* and Steven T. Boyce, Ph.D.*,†,‡,1

*Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio; †Department of Surgery, University of Cincinnati,Cincinnati, Ohio; and ‡Shriners Burns Hospital, Cincinnati, Ohio

Submitted for publication December 5, 2007

doi:10.1016/j.jss.2008.04.001

The Kerator is a computer controlled bioreactor forthe automated culture and harvest of keratinocytes thatcan reduce labor and materials involved in the fabrica-tion of engineered skin substitutes (ESS). Previous stud-ies have shown that the Kerator is comparable to tissueculture flasks by keratinocyte confluence during cul-ture, clonogenic potential of harvested keratinocytesand microanatomy, cell viability, and surface hydrationof ESS fabricated with the harvested keratinocytes. Inthis study, the Kerator and tissue culture flasks werefurther compared by keratinocyte proliferation in vitroand wound healing after transplantation of ESS to athy-mic mice. The number of bromodeoxyuridine-positivekeratinocytes in ESS fabricated with keratinocytes har-vested from Kerator after 2 wk of in vitro maturationwas 34 � 3 per high power field (hpf) (mean � SEM),which was not significantly different from that fabri-cated with keratinocytes harvested from flasks (34 � 1.5per hpf). Percentage original wound area 6 wk aftersurgery of ESS fabricated with keratinocytes from theKerator was 36% � 3.3%, which was not significantlydifferent from that of ESS fabricated with keratinocytesfrom flasks (30% � 4.3%). In both cases, 78% (7 of 9) micetransplanted were positive for engraftment of humankeratinocytes by direct immunofluorescence for HLA-ABC antigens. These results further confirm that theESS fabricated with keratinocytes harvested from Kera-tor and flasks are equivalent in vitro and in vivo. There-fore, use of Kerator for large scale production of ESS canlead to increased availability at reduced cost whilemaintaining ESS quality for grafting. © 2009 Elsevier Inc. All

rights reserved.

Key Words: automation; bioreactor; keratinocytes;engineered skin; wound healing

1 To whom correspondence and reprint requests should be ad-dressed at Department of Surgery, University of Cincinnati, P.O.

Box 670558, Cincinnati, OH 45267-0558. E-Mail: boycest@uc.edu

2960022-4804/09 $36.00© 2009 Elsevier Inc. All rights reserved.

INTRODUCTION

Skin is the largest organ in the body and provides abarrier against infections and fluid loss. Loss of thisprotective barrier contributes to the mortality andmorbidity due to burns involving a significant amountof total body surface area. Restoration of skin barrier isof definitive importance in the treatment of extensiveburn injuries, and this has been accomplished tradi-tionally by autograft or allograft. The limited availabil-ity of donor skin for autograft in large burns, and therisks of rejection and transmissible diseases associatedwith allograft, have led to the development of tissueengineered skin equivalents as alternatives in burnwound treatment. Various approaches have been takentoward the engineering of these therapeutic materials,including cultured epithelial autografts [1, 2] (Epicel);acellular dermal substitutes [3–6] (IntegraDRT, Allo-Derm); cell populated dressings [7] (TransCyte); cellu-lar dermal substitutes [8] (Dermagraft); and bilayeredskin substitutes [9, 10] (Apligraf, Orcel).

Engineered skin substitutes (ESS) are composed ofan epidermal substitute of autologous keratinocytes,attached to a dermal analogue of bovine collagen-glycosaminoglycan sponge populated with autologousfibroblasts. In vitro prior to grafting, ESS demonstratemorphogenesis similar to native human skin and forma functional epidermal barrier [11, 12]. After trans-plantation, ESS have stable engraftment, undergo his-togenesis, and heal wounds with minimal scarring [13].ESS reduce the requirement for skin autograft in thetreatment of massive burns and therefore demonstrateclinical efficacy as adjunctive therapy [14, 15]. Fabri-cation of ESS involves (1) isolation of keratinocytes andfibroblasts from a skin biopsy; (2) primary culture ofisolated cells; (3) cryopreservation of cells at the first

passage; (4) recovery from cryopreservation and inter-

297KALYANARAMAN AND BOYCE: IN VIVO EVALUATION OF KERATOR BIOREACTOR

mediate expansion of cells; (5) sequential inoculation ofexpanded fibroblasts and keratinocytes on collagen-GAG sponge; and (6) maturation of ESS at the air-liquid interface prior to grafting [11]. These processesare very intensive in terms of the labor and materialsused, which limits the amount of ESS that can befabricated at a given time, and also impacts the cost ofthe final product. As a means of increasing productavailability and reducing costs we have evaluated theKerator [16–18], a computer-controlled bioreactor, asan instrument for automation of keratinocyte expan-sion for fabrication of ESS [19].

The Kerator bioreactor was originally designed for theproduction of an autologous wound dressing composed ofsubconfluent keratinocytes attached to a transparent,gas permeable fluorinated ethylene propylene (FEP) film[17, 18]. To fabricate ESS with keratinocytes cultured inthe Kerator, a protocol for keratinocyte harvest was de-veloped to maintain the clonogenic potential of harvestedkeratinocytes in the subsequent passage. Subconfluentkeratinocytes in the Kerator were harvested by incubat-

FIG. 1. Schematic representation of the Kerator bioreactor,showing (A) growth chamber, (B) camera, (C) tilting actuator, (1) coldmedium reservoir, (2) warm medium reservoir, (3) cell seeding andcollection reservoir, (4) waste, (5) CO2 supply, (6) air supply, (7) massflow controller, (8) humidifier bottle, (9) CO2 detector, (10) Peltiercooler, (11) compressed air supply.

FIG. 2. Frontal view of the Kerator growth chamber in the hor(2) rocking platform, (3) pneumatic actuator, (4) camera, and (5) p

pneumatic actuator (3) allows medium and cells to be drained from the

ing in 0.02% ethylenediamine tetraacetic acid (EDTA) for8 to 10 min, followed by exposure to trypsin for 2 min.Keratinocytes harvested from the Kerator in this mannerhad colony-forming efficiencies and growth rates similarto those harvested from flasks, both with and withoutpretreatment with 0.02% EDTA. ESS fabricated withkeratinocytes cultured in, and harvested from, the Kera-tor were comparable in vitro to control ESS fabricatedwith keratinocytes harvested from standard tissue cul-ture flasks by histological organization, cellular viability,and surface hydration [19]. It is hypothesized, based onthese in vitro studies, that ESS fabricated with keratin-ocytes harvested from the Kerator will exhibit woundhealing comparable to those fabricated with keratino-cytes harvested from flasks, in vivo after transplantationto athymic mice. Demonstration of such comparabilitybetween Kerator and tissue culture flasks is an essentialstep toward the integration of the bioreactor in the pro-cess of ESS fabrication for clinical use, where the benefitsof automation on cost and availability of the therapeuticproduct can be fully realized. In this study, engraftmentof ESS prepared with normal human keratinocytes fromthe Kerator or flasks is compared.

MATERIALS AND METHODS

Kerator Bioreactor

The Kerator has been described in detail in earlier publications[16–19]. Briefly, it comprises of a modular polycarbonate growthchamber with FEP growth surface, connected to reservoirs of freshmedium, cell suspension, and waste by means of sterile siliconetubing. A bidirectional, multichannel peristaltic pump conveys me-dium and cells to and from the growth chamber. Fluid flow throughthe various tubes is regulated by means of pneumatic pinch valves(Fig. 1). The growth chamber rests on a platform that can be tilted to1 side by a pneumatic actuator (Fig. 2). The peristaltic pump, pinchvalves, and pneumatic actuator are controlled by a custom virtualinstrument (VI) designed in LabVIEW 6.1, which provides auto-mated fluid handling during medium changes and keratinocyte har-vest. A mass flow controller mixes air and 100% CO2 to provide 5%CO2 that is filtered and humidified before entering the growth cham-ber. The growth chamber is located in an insulated box in which thetemperature is maintained at 37°C by means of a hot plate and

ntal (A) and tilted (B) positions. (1) Five-layered growth chamber,matic pinch valves. Tilting the resting platform by activating the

izoneu

growth chamber. (Color version of figure is available online.)

298 JOURNAL OF SURGICAL RESEARCH: VOL. 152, NO. 2, APRIL 2009

thermostat. The Kerator is also equipped with a camera system thatallows real time visualization and monitoring of keratinocytes inculture.

Keratinocyte Culture and Harvest from Kerator

Normal human keratinocytes were inoculated in the Kerator at adensity of 4 � 103/cm2 of growth area. A custom VI was programmedto perform fully automated medium changes in the Kerator onceevery 48 h. The growth of keratinocytes was monitored by the onlineimage acquisition and analysis system. When the keratinocytes wereconsidered to be 75% to 90% confluent by image analysis (usually onthe 7th d of culture), the cells were harvested by treatment with0.02% EDTA for 8 to 10 min followed by brief (1 to 2 min) exposureto trypsin. The resultant cell suspension was collected in a reservoirof 10% fetal bovine serum, following which the growth chamber wasrinsed twice with HBS to collect the remaining cells. The cell sus-pension was centrifuged and the pellet was resuspended in modifiedMCDB 153. The cells were counted under a microscope using ahemacytometer and used for inoculation of ESS.

Tissue culture flasks were inoculated with keratinocytes at thesame density and medium was changed manually every 2 d. Whenkeratinocytes reached 75% to 90% confluence, they were harvestedby a 15-s rinse with HBS followed by exposure to trypsin for 5 min.The cell suspension was collected in 10% FBS and processed for ESSinoculation as described above.

ESS Fabrication

ESS were fabricated as described in earlier publications [12, 15],with syngeneic normal human fibroblasts, keratinocytes, and collagen-glycosaminoglycan sponge. Fibroblasts were inoculated on the spongeat a density of 0.5 � 106/cm2. The day following fibroblast inocula-tion, keratinocytes harvested from the Kerator or flasks were inoc-ulated on the sponge at a density of 1 � 106/cm2. The ESS wereincubated in the air-liquid interface for 3 wk following keratinocyteinoculation. UCMC 160 medium [20] was used for the first 3 d of ESSculture, and UCMC 161 medium was used for the remaining incu-bation period, with daily medium changes.

BrdU Immunofluorescence

The number of actively proliferating keratinocytes in the basallayers of ESS fabricated with keratinocytes harvested from Keratoror flasks was quantified by scoring of bromodeoxyuridine (BrdU)immunostaining of CSS in vitro. After 2 wks of maturation, test andcontrol ESS (n � 3) were incubated in 65 �M BrdU for 22 h at 37°Cand 5% CO2. Following incubation, ESS were fixed in formalin,embedded in paraffin, and sectioned. The sections were processed forBrdU immunostaining by baking at 60°C for 2 h followed by depar-affinization with xylene and rehydration with graded alcohols. Thesections were then co-labeled with 1:10 anti-BrdU fluorescein isocya-nate and 1:50 primary antipancytokeratin by a procedure describedpreviously. After labeling, the slides were washed with phosphate-buffered saline and Milli-Q water (Millipore Corporation, Bellerica,MA) and coverslipped with Vectashield hard mount media contain-ing 4=, 6=-diamidino-2-phenylindole. The slides were examined underthe microscope and 10 unique fields per ESS were analyzed for thenumber of BrdU positive cells (total 30 fields per condition). Data areexpressed as mean � SEM of BrdU positive cells per field.

ESS Transplantation to Athymic Mice

All animal care and handling in this study was performed accord-ing to protocols approved by the Institutional Animal Care and UseCommittee of the University of Cincinnati. ESS fabricated withkeratinocytes harvested from Kerator or flasks were allowed to ma-ture for 2 wk in vitro prior to transplantation on athymic mice

(nu/nu, Harlan; n � 9 animals per condition). Briefly, a full thickness

skin wound measuring 2 cm � 2 cm was prepared on the dorsolateralaspect of each mouse, sparing the panniculus carnosus. ESS alongwith an overlying piece of nonadherent dressing was placed ortho-topically on the wound and was secured to the wound margin withsutures. The grafted ESS were dressed with a piece of sterile gauzecoated with antibiotic ointment (containing Bactroban, Nystatin,and Neomycin in equal parts) and the gauze was held in place bytying the sutures over it. The grafted site was covered with a semi-occlusive dressing (OpSite, Smith & Nephew, Hull, United Kingdom)and the mice were wrapped in Coban (3M Corporation, St. Paul,MN). Mice were left undisturbed until 2 wk after the surgery whenthe dressings and sutures were removed. Thereafter, the mice weremaintained without dressings until 6 wk postoperative, at whichtime they were euthanized.

Wound Areas on Athymic Mice

Mice were photographed at biweekly intervals from 2 to 6 wk aftersurgery. The wound perimeters were traced at the time of surgeryand at weekly intervals from 2 to 6 wk postoperatively (n � 9 percondition at each time point). Wound area at each time point wasdetermined from these tracings using computer planimetry. Percentoriginal wound area was defined as the wound area at serial timepoints divided by the wound area at the time of surgery � 100%.Data for each time point were expressed as percent original woundarea (mean � SEM).

ESS Engraftment on Athymic Mice

Six weeks after surgery, the mice were euthanized and 2 biopsiesof the graft along with adjoining mouse skin were collected. Onebiopsy was processed by paraffin embedding and the other for cryo-microtomy. The paraffin embedded specimen was stained by H and Eand viewed under the microscope to analyze the histological organi-zation of healed tissue. The frozen sections were stained immuno-histochemically for HLA-ABC antigens by a procedure previouslydescribed [11, 12], to confirm the engraftment of human keratino-cytes on the mice. ESS engraftment was expressed as the percentageof animals staining positive for HLA-ABC (n � 9 each for Kerator orflasks).

Statistical Analysis

Data for wound area and ESS engraftment were analyzed by1-way repeated measures analysis of variance followed by Student-Neuman-Keul’s test for pair wise comparisons. Data for BrdU incor-poration were compared by Student’s t-test. Statistical significancewas accepted at the 95% confidence level (P � 0.05).

RESULTS

BrdU Immunostaining

Representative images of BrdU-positive keratino-cytes in ESS are shown in Fig. 3. In both conditions,the BrdU positive keratinocytes were located in thebasal layers of the epidermal portion of the ESS. Re-sults of the number of keratinocytes in ESS that werepositive for BrdU uptake d 14 of in vitro maturation issummarized in Fig. 4. There were no significant differ-ences between the Kerator (34 � 3 per hpf) and flasks

(34 � 1.5 per hpf).

sec

299KALYANARAMAN AND BOYCE: IN VIVO EVALUATION OF KERATOR BIOREACTOR

Wound Closure on Athymic Mice

Figure 5 shows representative animals grafted withESS fabricated with keratinocytes harvested from theKerator and flasks, at 2 and 6 wk after surgery. In bothconditions, the ESS attached to the wound and to thesurrounding margins of native mouse skin. The sur-faces of the grafted ESS were dry and well keratinized.ESS from both conditions displayed stable engraftmentover the 6-wk study period.

Wound Areas on Athymic Mice

Results of wound area measurements are illustratedin Fig. 6. The percent original wound area in micegrafted with ESS fabricated with keratinocytes har-vested from the Kerator was 83 � 6, 60 � 10, 36 � 3,35 � 3.2, and 36 � 3.3 at 2, 3, 4, 5, and 6 wk aftersurgery, respectively. At parallel time points, the per-cent wound area in mice grafted with ESS fabricatedwith keratinocytes harvested from flasks was 82 � 5.3,59 � 10.2, 40 � 4.1, 33 � 4.3, and 30 � 4.3. In bothconditions, the wound areas decreased significantly be-

FIG. 3. Representative images of ESS sections (viewed at 20�(B) flasks. BrdU positive nuclei stain fluorescent green and are locatthe epidermis are stained red by anti-pancytokeratin (see the Methods

FIG. 4. Number of BrdU-positive keratinocytes per high powerfield in ESS fabricated with keratinocytes harvested from Keratorand flasks, after 2 wk of maturation in vitro. There were no signifi-

cant differences between the conditions.

tween 2 and 4 wk (P � 0.05), but there was no changein wound area between 4 and 6 wk postop. There wereno significant differences in wound areas between the 2conditions at parallel time points.

ESS Engraftment on Athymic Mice

Representative H and E stained paraffin sections ofhealed ESS from test and control conditions are shownin Fig. 7A and C, respectively. Confirmation of engraft-ment of human keratinocytes was performed by directimmunofluorescence for HLA-ABC antigens. Positivestaining for HLA-ABC antigens in the epidermis ap-pears net-like, corresponding to the distribution ofthose antigens on the surface of keratinocytes. Thestaining is absent in the adjacent edge of normal mouseskin (Fig. 7B and D, arrow). Results of scoring forHLA-ABC immunostaining showed that in both condi-tions, 78% of animals (7 of 9) had keratinocytes stain-ing positive for HLA-ABC.

DISCUSSION

The Kerator bioreactor provides automation of ker-atinocyte culture [16–19] and harvest, thereby reduc-ing the labor and materials involved in the cell expan-sion phase of ESS fabrication. Data presented in thisstudy demonstrate comparability of ESS with keratin-ocytes from Kerator and flasks by BrdU incorporationin vitro, and wound healing after transplantation toathymic mice.

Previous studies have shown that ESS fabricatedwith keratinocytes harvested from Kerator and flasksare comparable in vitro by microanatomy, cell viabilityand surface hydration [19]. In this study, ESS from the2 conditions were compared in vitro by BrdU incorpo-ration in the epidermis as a measure of keratinocyteproliferation. No significant differences were found,validating the findings from earlier studies (Fig. 4).Also, it is known that the formation of a functional,barrier-forming epithelium both in vitro and in vivo

agnification) stained for BrdU-positive keratinocytes. (A) Kerator;redominantly in the basal layers of the epidermis. Keratinocytes in

tion). Scale bar � 100 �m. (Color version of figure is available online.)

med p

depends on a source of proliferative keratinocytes that

in

300 JOURNAL OF SURGICAL RESEARCH: VOL. 152, NO. 2, APRIL 2009

give rise to the mature corneocytes of stratum cor-neum. Because the BrdU studies on ESS were done atthe time of their transplantation, the results of thesestudies served as an indicator of their ability to healwounds effectively.

The athymic mouse has been the preferred model tostudy the wound healing capability of human skin sub-stitutes [21–33] because of its immunological toleranceof xenograft and the limited scarring that occurs dur-

FIG. 5. Representative images, at 2 and 6 wk after surgery, of afrom Kerator (A) and (B), or from flasks (C) and (D). The perimeterof wound contraction in both conditions. Also remarkable is the pconditions (arrows), suggesting the presence of human derived cells

FIG. 6. Percentage original wound area, of ESS fabricated withkeratinocytes harvested from Kerator and flasks transplanted onathymic mice. There were no significant differences between the 2

conditions at parallel time points.

ing healing. ESS grafts fabricated with keratinocytesharvested from the Kerator had well keratinized anddesquamating surfaces after 2 wk following surgery(Fig. 5). ESS have been shown to form a functionalepidermal barrier approaching that of native humanskin after transplantation to athymic mice [34, 35].Although surface hydration was not measured in vivoin the present study, the dry appearance of these graftsindicated well-formed epithelium. Evaluation of woundareas demonstrated that ESS fabricated with keratin-ocytes harvested from the Kerator heal wounds in amanner comparable to those fabricated with keratino-cytes harvested from standard tissue culture flasks(Fig. 6).

Engineered skin fabricated with keratinocytes fromflasks were shown to persist for as long as a year onathymic mice [36]. Tumorigenicity tests done by sub-cutaneous implantation of such cells in athymic miceruled out neoplastic transformation after 26-wk obser-vation [37]. ESS fabricated with keratinocytes har-vested form the Kerator showed stable integrationwith host skin for the 6-wk period of the present study.However long-term animal studies with these cells andESS fabricated with them will be necessary to test thestability of ESS. Although FEP has been used as cul-ture surface for growing cell types such as nerve [38]and corneal epithelial cells [39], use of the polymer forclinical transplantation of cells will require demonstra-tion of safety and purity which meet standards for

mic mice grafted with ESS fabricated with keratinocytes harvestedrafted area has been delineated with dashed lines. Note the extent

ence of pigmented spots within the grafted area at 6 wks in boththose regions. (Color version of figure is available online.)

thyof gres

Good Manufacturing Practices.

pid

301KALYANARAMAN AND BOYCE: IN VIVO EVALUATION OF KERATOR BIOREACTOR

Pigmented skin was seen within the graft region in 3out of 9 animals in both conditions, beginning from thefourth wk postop (Fig. 5B and D). This gave an earlyindication of the persistence of human-derived cellsbecause the skin of host animal is devoid of any pig-ment. This observation also suggests that the FEPculture surface may support the attachment and pro-liferation of melanocytes, which may be applicable forlarge scale culture of these cells in the Kerator.

Demonstration of comparability between the Kera-tor and flasks, both by in vitro and in vivo studies,suggests that the bioreactor can be used for automa-tion of keratinocyte culture for large scale fabricationof engineered skin. Apart from automation, the hands-free operation of cell culture facilitated by the Keratorlimits the chances of contamination [40]. By integrat-ing online monitoring sensors in the Kerator, it can bepossible to regulate medium changes according to pre-set thresholds of biochemical parameters. Such dy-namic bioprocess optimization has been shown to en-hance cell yield of hybridoma cells [41]. Adaptation ofthis technique to keratinocyte culture can prospec-tively lead to earlier achievement of an adequate cellyield for ESS fabrication, thereby reducing the leadtime between biopsy sourcing and clinical application.Metabolic regulation of keratinocyte culture in theKerator could also lead to improved cell physiology,thereby resulting in improved ESS quality that may

FIG. 7. Representative H and E stained sections and HLA-ABharvested from Kerator, (A) and (B), respectively, or from flasks (C)show normal mouse epidermis (to the right of arrow), which is thin a(to the left of arrow) resembles human skin and is derived from the g(D) shows net-like distribution of HLA-ABC (green) on human deristains negatively for HLA-ABC. Nuclei have been stained red by proonline.)

potentially translate into better wound healing in vivo.

Although analysis of cost involved in operating thebioreactor has not been performed, automation hashistorically resulted in greater product availability atlower cost and higher consistency. As more tissue en-gineered products become available commercially, bio-reactors, such as the Kerator, will be valuable tools inmaking them more available by the manufacturer, andmore affordable to the patient.

ACKNOWLEDGMENTS

The authors extend their gratitude to Andy Supp for assistancewith animal surgery; Jodi Miller for providing cell-culture media andassistance with animal surgery; and Chris Lloyd for fabrication ofcollagen sponges.

This project was supported by research grants to Dr. Boyce fromthe National Institutes of Health (GM050509) and Shriners Hospi-tals for Children (#8450).

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