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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, Japand 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 to15 years, cell-based therapies have in fact been clinically appliedfor 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 withsevere 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 appliedto restore tissue-specific functions and thereby avoid theneed for full organ transplantation. Similarly, the implantation of chondrocytes has also demonstrated the regeneration of hyalinecartilage in patients with full-thickness defects [22]. Results

from 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 widerange of ailments such as severe burns [23], skin ulcers [24], andgiant 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: [email protected] (T. Okano).

0168-3659/$ - see front matter 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2006.06.022
mailto:[email protected]://dx.doi.org/10.1016/j.jconrel.2006.06.022http://dx.doi.org/10.1016/j.jconrel.2006.06.022mailto:[email protected]


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stem cell deficiencies have also been developed [26]. Morerecently, various research groups have used epithelial grafts withvarious 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] , and the oral mucosal epithelium [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, thedevelopment 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 use

of 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) With

heterotypic 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


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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 the

cells 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, homotypiclayering 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 complex

laminar 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 by

dispase 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 ingrowth from the neighboring conjunctiva with neovascularization. (b) The conjunctival ingrowth is removedto re-expose the patient's corneal stroma. (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 then

carefully 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.)



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[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-thelial stem cells can be isolated and cultured on the temperature-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 comparison to cells harvested by treatment with dispase,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 autologous

oral 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.)



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transplantation, such as donor shortages and the risk of immunerejection, arise. To overcome these obstacles, we have alsodemonstrated that cell sheets composed of autologous oralmucosal epithelial cells can be used to treat these patients [63].Oral mucosal epithelial cell sheets fabricated on temperature-responsive dishes can be harvested and transplanted in the samemanner as corneal epithelial sheets ( Fig. 2). Interestingly, oralmucosal epithelial sheets fabricated by this method moreclosely resemble the native corneal epithelium than the nativeoral mucosal epithelium. Results from all cases in human trialshave demonstrated remarkably improved visual acuity, with allcorneas maintaining a clear and smooth surface ( Fig. 3). In arabbit model, follow-up results have also demonstrated that after transplantation, oral mucosal epithelial cell sheets undergochanges in their keratin expression profiles towards a corneal phenotype [64]. These findings therefore indicate that the direct interaction between the transplanted cell sheet with theunderlying corneal stroma may have an effect on the phenotypic

modulation of the oral mucosal epithelial cells.In addition to corneal surface reconstruction, we have alsodeveloped corneal endothelial cell sheets that closely resemblethe native endothelium [65,66] and are currently studying animalmodels for both corneal endothelial and retinal pigment epithelial cell sheets. Our use of carrier-free cell sheets is fa-vorable for ocular applications because it eliminates the need for additional substrates, scaffolds or sutures that may impair vision.

4. Myocardial tissue engineering

Recently the field of tissue engineering has garnered sig-nificant interest as a possible method to treat patients suffering

from severe heart failure. Various research groups have there-fore examined the use of various biomaterials, such as poly(glycolic acid) [67], gelatin [68], and alginate [69]; asscaffolding materials for the creation of myocardial tissue con-structs. Similarly, cardiomyocytes set in liquid collagen havealso been used to create engineered heart tissues [70,71] , withthe use of these tissue engineered constructs having been shownto improve cardiac function after transplantation to host hearts[68,69,72] . In contrast to these methods, we have designed anapproach for scaffold-free delivery of 3-D myocardial tissues.By layering cardiomyocyte sheets harvested from temperature-responsive dishes, beating cardiac tissues can be formed in vitro[57]. Upon stacking, these individual cell layers integrate toform a single, continuous, cell-dense tissue that resembles na-tive cardiac muscle. When these tissues were transplanted intothe backs of athymic rats, synchronous graft pulsations could beobserved macroscopically [57]. These implanted tissues showlong-term survival of more than 1 year, with resected grafts

demonstrating the presence of elongated sarcomeres, gap junc-tions and well-organized vascular networks within the bioengi-neered myocardium tissues [73].

Similarly, when layered cardiomyocyte sheets are transplanted directly to host hearts, they are able to form morphol-ogical connections to the heart with the presence of functionalgap junctions [74]. The transplantation of layered cardiomyo-cyte sheets was also able to repair damaged cardiac muscle, withimprovements in host ejection fraction observed after cell sheet transplantation [75].

In addition to cardiomyocyte sheet transplantation, we havealso demonstrated that the use of layered skeletal myoblast sheets can provide improved cardiac performance in diseased

Fig. 4. Endoscopic transplantation of autologous oral mucosal epithelial cell sheets to treat esophageal ulcerations. (a) After endoscopic submucosal dissection, anartificial ulceration is created in the lower esophagus. (b) The tissue engineered oral mucosal epithelial cell sheet is transferred to the ulcer site with the use of a whitesupport membrane. (c) Using endoscopic forceps, the cell sheet and the overlying support membrane are gently placed on the esophageal ulcer. (d) After transplantation, the forceps are withdrawn, leaving the cell sheet on the wound area. (e) An endoscopic mucosal resection tube is used to apply gentle pressure to the

cell sheet and the support membrane. (f) The cell sheet is then allowed to remain undisturbedat the transplant site for 15 min. (g) Using endoscopicforceps, the support membrane is carefully removed. (h) The autologous oral mucosal epithelial cell sheet remains stably attached to the esophageal ulcer site.



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cardiomyopathic animals [76]. Similarly, recent results withmesenchymal stem cell sheets have demonstrated improvedcardiac function after myocardial infarction [77]. The use of mesenchymal stem cell sheets was able to reverse cardiac wallthinning and improve animal survival after myocardial damage.Interestingly, cell growth of the transplanted mesenchymal stemcell sheets was observed after transplantation, with the differen-tiation of some stem cells into cardiomyocytes and blood ves-sels. Therefore with the development of cell delivery in the formof 3-D tissue structures created by cell sheet engineering, ad-ditional possibilities are now present for regenerative medicine.

5. Beyond simple transplantation of cell sheets

While we have outlined the basic approaches and providedexamples for cell transplantation using cell sheet engineering,new therapies are still required for the exploitation of effectiveregenerative therapies. In the following sections, we will discuss

the next generation of applications using cell sheet engineeringand the development of more advanced therapies.

6. Non-invasive endoscopic transplantation

In patients suffering from epithelial cancers of the gastroin-testinal tract, the development of endoscopic mucosal resection(EMR) has become a viable alternative to conventional opensurgery [78,79] . Recently, the development of a novel method of endoscopic submucosal dissection (ESD) has allowed for theremoval of large cancerous legions using a single operation,enabling precise diagnosis, as well as reducing the risk of recurrence [80,81] . In cases of esophageal ESD however, asignificant complication is the post-operative development of esophageal stenosis due to inflammation and scarring, that oc-curs during the natural progression of wound healing [82].

To overcome this problem, we have developed a new tech-nique involving ESD and endoscopic transplantation of

Fig. 5. Refractive surgery and epithelial cell sheet transplantation. Corneal epithelial cells were cultured for 2 weeks on temperature-responsive culture surfaces. After excimer laserphotoablation, the cell sheets are harvestedby temperature reduction to 20 C for30 minand transplanted to the ablatedstromal bed usinga donut-shapedsupport membrane. (Reprinted with permission from [91], 2006 Association for Research in Vision and Ophthalmology.)



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autologous oral mucosal epithelial cell sheets [83]. After ESD,these cell sheets that are created from a readily availableautologous cell source, can be transplanted with the use of endoscopic forceps and attach directly to the esophageal ulcer beds, without the need for sutures or other additional adhesiveagents (Fig. 4). The presence of an intact epithelium provided by the transplanted cell sheet dramatically enhances post-ESDwound healing and reduces host inflammatory responses.Therefore the use of endoscopic cell sheet transplantation also provides an additional approach for effective and non-invasivedelivery in cell therapies.

7. Combinatorial tracheal replacement

For the effective treatments of some diseases, cell sheet transplantation alone may not be sufficient for proper therapy or tissue reconstruction. In the case of tracheal replacement, the presence of a tubular support structure is required for the de-velopment of an effective prosthesis. Various synthetic, as wellas bioengineered, materials have therefore been applied asconduits for tracheal reconstruction [84 87]. However in these

cases, regeneration of the airway epithelium within the lumen of the prostheses is dependent on migration of cells from theneighboring portions of the host trachea [88,89] . Therefore, thelack of a well-developed epithelial layer can lead to problemsregarding graft patency after transplantation. For the develop-ment of a tracheal prosthesis possessing a mature pseudostra-tified epithelium, we transplanted tracheal epithelial cell sheetsinto the lumen of vascularized Dacron prostheses [90]. Four weeks after tracheal replacement, a mature epithelium could beobserved throughout the grafts that had received cell sheet transplantation. In contrast, control prostheses possessed only athin and immature epithelial layer at the center of the trachealgrafts. Within the lumen of the trachea, there are significant shear forces and stress due to normal respiratory functions. Therefore

the ability for cell sheets to form stable attachment and remainwithin the lumen, is a key factor in the rapid regeneration andmaintenance of a mature epithelium. The effectiveness of acombinatorial approach using traditional tissue engineering approaches with cell sheet engineering therefore also providessignificantly new avenues for the reconstruction of more complex tissues.

8. Refractive surgery and cell sheet transplantation

Another area where we have applied cell sheet engineering ina novel manner is for refractive surgery [91]. Traditional meth-ods such as photorefractive keratectomy (PRK) and photother-apeutic keratectomy (PTK) apply the use of excimer laser photoablation to the corneal surface for the removal of super-ficial opacities or for the correction of visual acuity [92 94].However, a major concern is the post-operative development of corneal haze which can have significant consequences on visual

Fig. 6. Polysurgery to create thick, vascularized myocardial tissues. (a) Individual cardiomyocyte sheets are harvested from temperature-responsive culture dishes andstacked to create layered constructs. (b) Triple-layer myocardial grafts are transplanted subcutaneously. (c) After transplantation, neovascularization occurs within the bioengineered tissues. (d) After sufficient neovascularization has occurred, a second triple-layer myocardial graft is transplanted directly over the first construct. (e) Neovascularization of the second graft occurs through the first construct, creating thick tissues that can overcome the limits of passive diffusion. (Reprinted with permission from [106], 2006 Federation of American Societies for Experimental Biology.)

Fig. 7. Engineering of 1 mm thick myocardial tissues using polysurgery. Using10-times polysurgery, approximately 1 mm thick synchronously beating

myocardial tissues can be engineered in vivo. (Reprinted with permission from[106] , 2006 Federation of American Societies for Experimental Biology.)



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outcomes [95 97]. To prevent the development of haze, topicalapplication of the anti-cancer drug mitomycin C is commonlyused [98 100] , however the effect of mitomycin C induced celldeath in normal tissues cannot be excluded. Recently, laser insitu keratomileusis, commonly known as LASIK, has beenapplied, where a corneal flap is created with laser ablation performed on the underlying corneal stroma [101]. However,several flap-related complications can occur after deep ablationsand LASIK can only be used in the correction of slight refractiveerrors [102 104] . Therefore, methods to inhibit the develop-ment of corneal haze after refractive surgery are a key factor inenhancing the post-operative outcome. During normal refractivetherapies, the use of the excimer laser results in the completeremoval of a portion of the corneal epithelium and underlyingstroma to an established depth. Post-operatively, re-epithelial-ization of the ablated regions occurs by the migration of neighboring epithelial cells to cover the exposed stromal bed.Previously the causes of corneal haze have been correlated to

the processes that occur at the interface between the stroma andthe newly-formed epithelium of ablated stromal bed [105]. Wetherefore sought to enhance post-operative healing by trans- planting corneal epithelial cell sheets immediately after excimer laser photoablation. The tissue engineered epithelial cell sheetscan attach even to the ablated portions of the corneal stromal bedwithout the use of sutures or adhesive agents, providing an intact epithelial barrier immediately following laser keratectomy(Fig. 5). Using this method, results showed significantly de-creased corneal haze both 1 and 2 months after surgery, as well asa reduction in the number of inflammatory cells and activatedfibroblasts within the corneal stroma [91]. These results showedan effective use of cell delivery after conventional laser kera-tectomy to decrease post-operative haze. We are currently plan-ning the application of these methods for human patients in thenear future.

9. Polysurgery to create thick 3-D tissues

While we have previously demonstrated the reconstruction of 3-D myocardial tissues via the layering of cardiomyocyte sheets,the limits of passive diffusion that restricts both the supply of nutrients and the removal of metabolic wastes limits the viabletissue thickness of all engineered constructs. Therefore, evenwith the layering of additional cell sheets, limits on tissue

thickness are approximately 100 m, which prevents the un-limited stacking of cell sheets in the creation of thicker tissueconstructs [106]. To overcome this restriction, we have devel-oped a method of polysurgery which applies the use of repeatedtransplantations after allowing for intervals in which sufficient neovascularization has occurred ( Fig. 6) [106]. Using thismethod, triple-layer cardiomyocyte sheets were transplantedsubcutaneously 10 times, at intervals of 1 day to produce approximately 1 mm thick, cell-dense myocardial tissues ( Fig. 7).These thick tissues showed synchronous pulsations as well asthe development of a well-organized microvascular network.However as this method of repeated transplantations carries arelatively high risk of complications if applied directly to thehost heart, approaches to ectopically fabricate thick tissues are of

significantly higher appeal. By applying this method of polysurgery over a large artery and vein, thick tissue constructswith connectable vessels could be created and used for ectopictransplantation, without the need for surgically resecting and re-connecting numerous microvessels, which is technically im- possible. When thick tissue grafts were fabricated over thecaudal epigastric artery and vein, blood supply to the engineeredtissue was completely supplied from the underlying vessels.After ectopic transplantation and anastomosis to the carotidartery and jugular vein in the neck of another athymic rat, thegrafts showed continuous pulsation and survival for more than1 month [106].

The use of polysurgery therefore allowed for the re-creationof thick tissues, far exceeding the limits of mass transport andcan likely be applied to other tissues. We are also currentlyinvestigating other methods for the creation of thicker tissuesunder in vitro conditions.

10. Conclusions

While the fields of regenerative medicine and tissueengineering have made significant advances over the past 20 years, there still remains considerable difficulty in re-creatingmany tissues and organs, due to the limitations of traditionalmethods. Herein, we presented the applications of cell sheet engineering using temperature-responsive culture dishes as anovel approach for the delivery of both two- and three-dimen-sional cell-based constructs. We believe that the various applications of cell sheet engineering will overcome the problemsthat have limited conventional approaches and establish a new basis for cell-based therapies.

Acknowledgements

This work was supported in part by the Center of Excellence(COE) Program for the 21st Century and the High-TechResearch Center Program, from the Ministry of Education,Culture, Sports, Science and Technology (MEXT), Japan; theCore Research for Evolution Science and Technology Program(CREST) by the Japan Science and Technology Agency (JST);the Core To Core Program from the Japan Society for thePromotion of Science (JSPS); and the Suzuken MemorialFoundation.

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