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Cell Delivery Using Cell Sheet

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  Cell delivery in regenerative medicine: The cell sheet engineering approach Joseph Yang  a  , Masayuki Yamato  a  , Kohji Nishida  b , Takeshi Ohki  c , Masato Kanzaki  d ,Hidekazu Sekine  a  , Tatsuya Shimizu  a  , Teruo Okano  a, ⁎ a   Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan  b  Department of Ophthalmology and Visual Science, Tohoku University Graduate School of Medicine, Miyagi, Japan c  Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, Tokyo, Japan d  Department of Surgery I, Tokyo Women's Medical University, Tokyo, Japan Received 1 June 2006; accepted 21 June 2006Available online 27 June 2006 Abstract Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success inclinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, wehave developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells asintact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without theuse of scaffolding or carrier materials, or used to create  in vitro  tissue constructs via the layering of individual cell sheets. In addition to simpletransplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorialtissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.© 2006 Elsevier B.V. All rights reserved.  Keywords:  Temperature-responsive culture surface; Tissue engineering; Transplantation 1. The development of regenerative medicine While the related fields of regenerative medicine and tissueengineering have generated marked interest over the past 10 to15years,cell-basedtherapieshaveinfactbeenclinicallyappliedfor nearly 40 years [1,2]. Over this period of time, direct injection of bone marrow cells has been used in the treatment of  both malignant  [3 – 5] and non-malignant diseases [6 – 9]. Celltransplantation has also been examined in attempts to restoreneuronal function in patients suffering from Parkinson's disease[10] and other neural disorders [11], to rescue patients with severe liver failure [12 – 14], and for therapeutic angiogenesis in patients suffering from limb ischemia [15 – 17]. Recently,injection of bone marrow cells [18] and skeletal myoblasts[19 – 21] directly to ischemic hearts have been clinically appliedtorestoretissue-specificfunctionsandtherebyavoidtheneedfor full organ transplantation. Similarly, the implantation of chondrocytes has also demonstrated the regeneration of hyalinecartilage in patients with full-thickness defects [22]. Resultsfrom these previous experiences have therefore convinced re-searchers that cell transplantation can be a suitable treatment for a variety of diseases that occur in a wide range of tissue systems.Aside from the direct injection of single cell suspensions, thedevelopment of tissue engineering has also become a novelavenue for regenerative therapies. Since the early 1980s, epi-dermal grafts have been created by the  in vitro  culture of keratinocyte stem cells isolated from small biopsies. Thesemethods have been used to treat patients suffering from a widerangeof ailments suchas severe burns [23],skin ulcers[24],and giant congenital nevi [25]. Using similar methods, cornealepithelial grafts to treat patients suffering from corneal epithelial Journal of Controlled Release 116 (2006) 193 – 203www.elsevier.com/locate/jconrel ⁎  Corresponding author. Institute of Advanced Biomedical Engineering andScience, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku,Tokyo 162-8666, Japan. Tel.: +81 3 3353 8111x30233; fax: +81 3 3359 6046.  E-mail address:  tokano@abmes.twmu.ac.jp (T. Okano).0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2006.06.022  stem cell deficiencies have also been developed [26]. Morerecently,variousresearchgroupshaveusedepithelialgraftswithvarious carrier materials such as amniotic membrane [27 – 30],collagen gels and matrices [31], and fibrin gel [32 – 34] to createconstructs for the regeneration of the epidermis [32,35], cornealepithelium[27 – 31,33,34],andtheoralmucosalepithelium[36]. Tissue engineering using conventional methods of seedingcells into biodegradable scaffolds has also recently yieldedclinical products, with applications for bone [37], cartilage [38],  blood vessels [39,40], and heart valves [41]. Most recently, the development of autologous tissue engineered bladders have been shown to be effective in treating patients with end-stage bladder disorders [42]. These results have therefore signaled thearrival of regenerative therapies utilizing techniques more in-volved than simple cell injection. 2. Cell sheet engineering Recently we have developed an alternative method for regenerative therapies by applying the use of temperature-responsive culture dishes [43,44]. The temperature-responsive polymer poly(  N  -isopropylacrylamide) (PIPAAm) undergoes adistinct transition from hydrophobic to hydrophilic across itslower critical solution temperature (LCST) of 32 °C [45].Therefore, by covalently immobilizing PIPAAm onto ordinarytissue culture polystyrene (TCPS) surfaces at nanometer-scalethickness, control of cell adhesion and detachment can becontrolled by simple temperature changes [46]. On thesesurfaces, various cell types adhere, spread and proliferatesimilarly to on normal TCPS at 37 °C. However, by reducingthe incubation temperature to 20 °C, all cultured cellsspontaneously detach due to the conversion of the graftedPIPAAm from hydrophobic to hydrophilic. Utilizing thesetemperature-responsive surfaces, we have developed a strategyof   “ cell sheet engineering ”  whereby cultured cells are harvestedas intact sheets along with their deposited extracellular matrix(ECM) and can be used for tissue engineering applications. Incontrast to previous methods, the use of temperature-responsivesurfaces allows for cultured cells to be harvested without the useof proteolytic enzymes such as trypsin or dispase which canresult in cell damage and loss of differentiated phenotypes[47,48]. Additionally, as ECM remains present on the basal Fig. 1. Cell-based regenerative therapies using cell sheet engineering. (a) Using direct transplantation of single cell sheets, skin epidermis, corneal epithelium, bladder urothelium and periodontal ligaments can be reconstructed. (b) By homotypic layering of cell sheets, pulsatile myocardial tissues can be re-created. (c) Withheterotypic stratification of various cell sheets, higher order laminar structures such as liver lobules and kidney glomeruli can be engineered. (Reprinted with permission from [59], © 2005 Elsevier.)194  J. Yang et al. / Journal of Controlled Release 116 (2006) 193  –  203  surface of the cell sheets [49], they can be directly transplantedto tissue beds or even layered to create three-dimensional (3-D)tissue-like structures.This approach therefore seemingly provides several advan-tages over traditional regenerative therapies of cell injection andtissue reconstruction with biodegradable scaffolds. With the useof single cell suspensions, there is often a significant loss of cells, with only a small percentage of cells remaining at the siteof interest. In addition, in cases of damaged tissues, injectedcells are often unable to attach at sites where the host archi-tecture is destroyed. In contrast, cell sheets via their depositedECM, can be attached to host tissues and even wound sites, withminimal cell loss.While polymer scaffolds have been applied for the recon-struction of some tissues, a key concern is the inflammatoryreactions that occur upon their implantation and biodegradation[50]. While degradation can result in integration of theengineered tissue constructs, it can also cause damage to thecells seeded within the scaffolds. Additionally, although therehas been success in the reconstruction of some tissues such as bone and cartilage, the use of biodegradable scaffolds oftencannot adequately reproduce the cell density that is required of other tissues such as the liver, heart or kidney.As the detailed architecture of many tissues can be thought of as consisting of densely packed cells with little ECM, thereconstruction of the tissues using cell sheets can therefore be performed in three general ways. First, single cell sheets can bedirectly transplanted to host tissues as in the cases of skin [51],corneal epithelium [52], bladder urothelium [53,54], and  periodontal ligaments [55,56] (Fig. 1a). Second, homotypic layering of cell sheets can be used to re-create 3-D structuressuch as cardiac muscle [57,58] (Fig. 1 b) and smooth muscle [59]. Finally, by stratifying different cell sheets, more complexlaminar structures such as liver lobules [60] and kidneyglomeruli can be constructed (Fig. 1c). Using these approaches,cell sheet engineering therefore allows for cell delivery whileavoiding the use of single cell injection or biodegradable scaf-folds. In the following sections, we will describe some of thevarious methods for cell delivery and tissue reconstructionusing cell sheet engineering. 3. Corneal surface reconstruction Following the success of epidermal tissue engineering,corneal epithelial tissue engineering has emerged in 1990s toovercome the problems of immunorejection of transplantedtissues as well as donor organ shortages [61]. In cases of cornealepithelial disorders due to severe disease or trauma, thecomplete loss of corneal epithelial stem cells results in thedevelopment of corneal opacity, followed by loss of visualacuity. Using cultured corneal epithelial stem cells harvested bydispase treatment, Pellegrini et al. showed the recovery of corneal transparency and improved visual acuity in patientsreceiving autologous corneal epithelial grafts [26]. Morerecently, to overcome the fragility of epithelial grafts harvested by dispase treatment, several investigators have describedtransplantation of corneal epithelial cells expanded  ex vivo  onvarious carrier substrates such as amniotic membrane [27 – 30],collagen gel [31] and fibrin gel [33,34]. Using these methods, the resultant constructs composed of both epithelial cells and thecarrier are then transplanted onto the corneal stroma and held in place by sutures. However, although the use of carrier substratesallows for easy handling of the engineered constructs, their  presence can potentially influence the post-operative clinicaloutcomes. After transplantation, amniotic membrane persists between the corneal stroma and the expanded epithelial cells Fig. 2. Corneal reconstruction using tissue engineered cell sheets composed of autologous oral mucosal epithelium. (a) Pre-operatively, the patient's corneal surface iscoveredby ingrowthfrom the neighboring conjunctiva withneovascularization. (b) The conjunctival ingrowthis removedtore-expose the patient's cornealstroma.(c)With the use of a donut-shaped support membrane, the oral mucosal epithelial cell sheet is harvested from the temperature-responsive culture surface. (d) The tissueengineered autologous epithelial cell sheet is placed directly on the patient's corneal stroma and attaches stably after 5 min. (e) The donut-shaped supporter is thencarefully removed. (f) The cell sheet provides an intact epithelial barrier forming a clear and smooth ocular surface. (Reprinted with permission from [63], © 2004Massachusetts Medical Society. All rights reserved.)195  J. Yang et al. / Journal of Controlled Release 116 (2006) 193  –  203  [62], and can also have an effect on the optical transparency of the fabricated constructs. Conversely, while fibrin gel degradesrapidly after transplantation to the corneal stroma, a strict re-quirement in applications to the ocular surface is that the carrier completely resolves without any scarring. Therefore theresultant inflammation due to fibrin biodegradation may possibly result in microtrauma to the corneal stroma whichcan have an effect on post-operative outcomes. In addition, the possibility of infection from the use of biological carries such asanimal-derived collagen, human blood-derived fibrin, andamniotic membrane cannot be completely excluded. Due tothese concerns, an ideal approach for ocular applicationstherefore seems to involve the creation of carrier-free constructsthat can be easily manipulated during surgical operations.For the reconstruction of the corneal epithelium, limbal epi-thelialstemcellscanbeisolatedandculturedonthetemperature-responsive culture surfaces at 37 °C. After harvest by tem- perature reduction to 20 °C for 30 min, these cell sheets alongwith their deposited ECM can be easily manipulated and easilyadhere to the host corneal stroma without the need for sutures[52]. In comparisonto cells harvested by treatment withdispase,corneal epithelial cell sheets fabricated on temperature-respon-sive culture dishes are less fragile and contain both cell-to-cell junction and ECM proteins that can be damaged by dispase. Inthis way, a well-formed epithelial sheet can be transplantedwithout the need for any carrier substrate, such as amnioticmembrane or fibrin gel. After 5 min, the transplanted cell sheetsattach to the corneal stroma without the need for sutures. In patients receiving corneal epithelial cell sheet transplantation,the corneal surface remains clear with significantly improvedvisual acuity, more than one year after surgery.However, in cases of severe disease, damage to both eyes prevents the use of autologous corneal epithelial stem cells andtherefore the problems associated with traditional corneal Fig. 3. Clinical outcomes of ocular surface reconstruction using autologous oral mucosal epithelial cell sheets. Photographs taken just before grafting of autologousoral mucosal epithelial cell sheets and post-operatively at 13, 14, or 15 months. Results are presented for the first four consecutive patients enrolled in the study.(Reprinted with permission from [63], © 2004 Massachusetts Medical Society. All rights reserved.)196  J. Yang et al. / Journal of Controlled Release 116 (2006) 193  –  203
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