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BACKGROUND: Bone marrow mesenchymal stem cells have good potential for directional differentiation, but the effect and mechanism of enamel matrix derivatives on osteogenic differentiation are still unclear. OBJECTIVE: To summarize the latest research progress on osteogenic induction of bone marrow mesenchymal stem cells by enamel matrix derivatives. METHODS: Related literature from 2000 to 2020 was searched in CNKI, Wanfang Data, VIP Database, PubMed databases. The key words are “Emdogain® or enamel matrix derivatives, bone marrow mesenchymal stem cells”. The languages of the literature were set to Chinese and English. When retrieving some classic articles, the publication date could be extended appropriately. Finally, 62 English articles and 5 Chinese articles meeting the inclusion criteria were selected. RESULTS AND CONCLUSION: There are different reports on the osteogenic effect of enamel matrix derivatives on bone marrow mesenchymal stem cells. Enamel matrix derivatives may enhance the osteogenic induction ability of bone marrow mesenchymal stem cells, and may enhance the osteogenic effect by affecting cells or cell membranes, but the relevant mechanism is unclear.
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Scaffold-free tissue engineered cell sheet is an emerging technology in biomedical field. It can avoid the adverse effects of scaffold materials, and can be further assembled to form more complex three-dimensional functional tissues. The construction of cell sheet is mainly based on the culture substrate composed of sensitive materials. By changing the stimulation factors such as temperature, enzyme, light, ion, redox, pH and sugar, the adhesion behavior of the substrate to the cells could be changed to make the cells detach naturally, thus generating the cell sheet. Recent years have seen the development of various simple and efficient construction technologies of cell sheet due to the development of a variety of novel sensitive culture substrates. The resulted cell sheets with excellent performance have greatly expanded their applications. This review summarized the construction methods of tissue engineered cell sheet and discussed the challenges and future perspectives in this field.
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Temperature , Tissue Engineering , Tissue ScaffoldsABSTRACT
Oromaxillofacial hard tissue defects is still a difficult problem in clinical treatment. Regeneration of oromaxillofacial hard tissue based on tissue engineering technology has a good clinical application prospect. The functional modification of scaffolds is one of key factors that influence the outcome of tissue regeneration. The biomimetic design of biomaterials through simulating the natural structure and composition of oromaxillofacial hard tissue has gradually become a research hotspot due to its advantages of simplicity and efficiency. In this article, the biomimetic modification of biomaterials for oromaxillofacial hard tissue regeneration is reviewed, expecting to provide a new idea for the treatment of oromaxillofacial hard tissue defect.
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Biocompatible Materials , Biomimetics , Bone Regeneration , Dental Implants , Tissue Engineering , Tissue ScaffoldsABSTRACT
BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) are a kind of adult stem cells that can be extracted from discarded placenta. Compared with other mesenchymal stem cells, hAMSCs have many advantages such as noninvasive isolation, low immunogenicity and short growth cycle, and thus hAMSCs are an important source of tissue engineering seed cells. Currently, hAMSCs have been applied in the clinical treatment of diabetes. OBJECTIVE: To explore a simple method to fabricate hAMSC sheets and to study their potential to differentiate into osteocytes. METHODS: Passage 3 hAMSCs were seeded into culture plates at a high density, and the sheet-forming medium was added to fabricate hAMSC sheet. The structural characteristics of hAMSC sheets were evaluated by histological staining and scanning electron microscopy. The sheet-forming induction medium was added to the passage 3 hAMSCs for 7 continuous days, and then replaced by the osteogenic induction medium for 14 days to construct osteogenic-induced hAMSC sheets. The potential for osteogenic differentiation of hAMSC sheets was assessed by alizarin red staining, immunohistochemical staining, alkaline phosphatase activity, RT-PCR, and western blot assay. RESULTS AND CONCLUSION: Hematoxylin-eosin staining results indicated that hAMSC sheets had a multi-layered structure with the cells stacked layer-by-layer and evenly distributed. Under the scanning electron microscope, the hAMSC sheets had a multi-layered structure, and a large amount of extracellular matrices that enveloped the fusiform cells were produced. After 14 days of osteogenic induction, orange-red precipitation was observed by alizarin red staining. Immunohistochemical staining results showed a large amount of type II collagen. Compared with non-induced hAMSC sheet, alkaline phosphatase activity was significantly increased in the osteogenic induced sheet (P < 0.01). The expression levels of type I collagen, osteocalcin, and Runt-related transcript factor 2 mRNA and protein were significantly higher in the osteogenic-induced hAMSC sheet group than the non-induced hAMSC sheet group (P < 0.05). Our findings indicate that this is a simple and economic method to construct the hAMSC sheets in the normal culture medium. Moreover, hAMSC sheets have a good osteogenic differentiation potential that has been confirmed in vitro.
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@#At present, conventional periodontal treatment cannot achieve complete and effective periodontal tissue regeneration. Cell sheet technology (CST) is a kind of cell transplantation method without scaffold material that can maintain complete extracellular matrix, important ion channels of cells, growth factor receptors, etc., and ensure the interaction between cells and the extracellular matrix. In this paper, the application and research progress of the cell sheet in the field of periodontal tissue regeneration are reviewed. Different types of seed cells can be prepared into monolayer cell sheet, multilayer cell sheet, cell sheet fragments and cell sheet polymers. Among them, the monalayer cell sheet is easily damaged and requires high deoperator; the multilayer cell sheet shows improved mechanical properties, but its thickness needs to be controlled to avoid cell necrosis. The cell sheet fragment can be used in the narrow space between the alveolar bone and root cementum to reduce the difficulty of operation and improve the mechanical properties of the cell sheet. Cell sheet polymers are three-dimensional structures that can provide strong mechanical support and improve the stability of the cell sheet, but the stability of their biological activity needs to be further improved. In methods for construction of the cell sheet, the antifibrosis and antiangiogenesis properties of the amniotic sheet have shown that this structure is suitable as the matrix of cell culture; the method of using a temperature-sensitive culture dish is simple and easy; continuous induction with vitamin C can retain some important proteins on the cell surface; and the magnetic tissue engineering method can increase cell adhesion and easily form a stable cell sheet. The above methods have their own characteristics. In clinical applications, monolayer cell sheet is mainly used for direct transplantation to the receiving site to construct periodontal tissue; multilayer cell sheet of the same or different species overlap and are then transplanted to the receiving site; and multilayer cell sheet of the same kind are wrapped with scaffold material and then transplanted to the receiving site to construct a three-dimensional structure. Overall, cell sheet technology has shown good potential in periodontal tissue regeneration.
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Objective: To explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology. Methods: Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression. Results: HDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a "honeycomb" network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant ( P>0.05). Conclusion: Three-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.
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BACKGROUND: Engineered cell sheet transplantation has been considered an alternative physiological therapy for endocrine disorders. In this study, we attempted to fabricate functional human thyroid cell sheets using the engineering technology by culturing primary thyrocytes in free-feeder monolayers and assessed their proliferation and function in two different media. METHODS: The non-tumorous tissues (approximately 2 g) were dissected during surgery. Primary human thyroid cells were isolated by mechanical dispersion and treatment with isolation solution. The cells were cultured on tissue culture dishes or temperature-responsive culture dishes to induce the formation of detached cell sheets. RESULTS: Primary thyroid cells isolated from nine patients were positive for thyroid transcription factor 1, thyroglobulin (TG) and cytokeratin 7. Cell sheets with follicles were fabricated by cells incubated in both Dulbecco's Modified Eagle Medium (DMEM) and hepatocyte-defined medium (HDM) culture medium. The diameter and thickness of sheets fabricated in HDM were larger and thicker than those fabricated from DMEM. Furthermore, the cells incubated in HDM secreted higher levels of fT3 and fT4 than those incubated in DMEM. The thyroid peroxidase and TG mRNA of cells maintained in HDM were higher than those in cells maintained in DMEM. CONCLUSION: HDM appears suitable as a culture medium for maintaining primary thyrocytes and fabricating functional cell sheets. These in vitro findings may contribute to the development of appropriate culture conditions for human thyrocytes as well as engineered functional cell sheets.
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Humans , Eagles , In Vitro Techniques , Iodide Peroxidase , Keratin-7 , RNA, Messenger , Thyroglobulin , Thyroid Gland , Transcription FactorsABSTRACT
Objective: To construct a novel tissue engineering complex, BMSCs sheet-RADA16 scaffold, by combining cell sheet and self-assembled peptides.. Methods: The self-assembled peptide RADA16 scaffold was wrapped with the BMSCs cell sheet. The morphology of the cells and the complex were observed by SEM and confocal laser microscopy, and the proliferation of cells was assessed by CCK-8. The osteogenic differentiation of BMSCs was examined by detection of related gene expression with RT-PCR. Results: Compared with the BMSCs cell sheet, the numbers of cells on RADA16 scaffold growth rapidly at 3 rd-8 th day, and BMSCs were more on the scaffold than those on the cell sheet (P＜0. 05) . RT-PCR results showed that the expression level of osteogenesis-related genes was higher in the complex (P＜0. 05) . Conclusion: BMSCs Sheet-RADA16 Scaffold may promote proliferation and osteogenic differentiation of BMSCs.
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The loss of periodontal support tissues might cause movement or finally loss of the teeth affected, impair furthermore the pronunciation and mastication functions, and even result in a series of physiological and psychological problems. Tissue engineering, as a technology to remodel missing tissues or organs and functional reconstruction, has achieved gratifying progress in regeneration of periodontal tissues. However, conventional construction methods have some deficiencies for functional periodontal reconstruction. In recent years, with the progress of tissue engineering technology, a series of new techniques and methods, such as cell sheet technology, decellular technology, electrospinning technology and three-dimensional printing, has been applied in tissue engineering bringing new hope for the regeneration of periodontal tissues. In this review article, the recent progress achieved in the field of periodontal tissue engineering and application of modern technology are summarized to make a brief exposition and to explore the future development of periodontal regeneration.
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The purpose of this study was to evaluate the osteogenesis ability of osteogenic matrix cell sheets (OMCS) derived from old donor cells. Bone marrow stromal cells (BMSC) were obtained from young (7-week-old) and old (1-year-old) Fischer344 rats donors and cultured with modified Eagle's medium (MEM group) alone or containing dexamethasone (Dex; 10 nM) and ascorbic acid phosphate (AscP; 0.28 mM) (Dex/AscP group). We prepared four in vitro experimental groups: (1) young MEM, (2) young Dex/AscP, (3) old MEM and (4) old Dex/AscP. Cell proliferation and osteogenic marker mRNA expression levels were evaluated in vitro. To assess bone formation in vivo, the cells of each group were combined with beta tricalcium phosphate (TCP) disks followed by implantation in recipient rats. The in vitro study showed significant differences in the mRNA expression of osteocalcin, ALP, and BMP2 between MEM and Dex/AscP groups. Bone formation following implantation was observed upon histological analyses of all groups. TCP combined with OMCS (OMCS/TCP group) resulted in enhanced bone formation compared to that following combination with BMSC (BMSC/TCP). The osteocalcin content of the OMCS/TCP group 4 weeks after implantation was significantly higher than that in the BMSC/TCP construct for both young and old donors. The present study clearly indicated that OMCS could be generated from BMSCs of old as well as young donors using a mechanical retrieval method. Thus, through its usage of OMCS, this method may represent a potentially effective therapeutic option for cell-based therapy in elderly patients.
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Aged , Animals , Humans , Rats , Ascorbic Acid , Bone and Bones , Bone Marrow , Cell Proliferation , Dexamethasone , In Vitro Techniques , Mesenchymal Stem Cells , Methods , Osteocalcin , Osteogenesis , RNA, Messenger , Tissue DonorsABSTRACT
Objective: To review the progress of cell sheet technology and its application in bone and cartilage engineering.
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The optimal cell culture method of autologous oral mucosal epithelial cell sheet is not well established for a safe transplantation on to the patients' ocular surface. Animal serum and 3T3 mouse feeder cells are currently being used to stimulate the growth of the epithelial cells. However, the use of animal compounds can have potential side effects for the patient after transplantation of the engineered cell sheet. In the present study, we focused on engineering a rabbit oral mucosal epithelial cell sheet without 3T3 mouse feeder cells using a mix of Dulbecco's Modified Eagle Medium/Bronchial Epithelial Cell Growth Medium culture media (DMEM/BEGM). Autologous oral mucosal epithelial cell sheets, engineered with DMEM/BEGM feeder cell free culture media, were compared to those cultured in presence of serum and feeder cells. Using a DMEM/BEGM mix culture media, feeder cell free culture condition, autologous oral mucosal epithelial cells reached confluence and formed a multilayered sheet. The phenotype of engineered cell sheets cultured with DMEM/BEGM were characterized and compared to those cultured with serum and feeder. Hematoxylin and eosin staining showed the formation of a similar stratified multilayer cell sheets, in both culture conditions. The expression of deltaN-p63, ABCG2, PCNA, E-cadherin, Beta-catenin, CK3, CK4, CK13, Muc5AC, was similar in both culture conditions. We demonstrated that rabbit autologous oral mucosal epithelial cell sheet can be engineered, in feeder cell free conditions. The use of the DMEM/BEGM culture media to engineer culture autologous oral mucosa epithelial cell sheet will help to identify key factors involved in the growth and differentiation of oral mucosal epithelial cells.
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Animals , Humans , Mice , beta Catenin , Cadherins , Cell Culture Techniques , Culture Media , Eagles , Eosine Yellowish-(YS) , Epithelial Cells , Feeder Cells , Hematoxylin , Methods , Mouth Mucosa , Phenotype , Proliferating Cell Nuclear AntigenABSTRACT
Objective To probe the periodontal ligament regeneration following the implantation of bone marrow mesenchymal cells ( BMSCs ) sheet-collagen membrane-BMSCs sheet sandwich complex.Methods BMSCs cell sheet-collagen membrane-BMSCs sheet complexes were compounded on the root surface of teeth of Beagle dogs.All the dogs were killed on 4 and 12 weeks after implantation.Periodontal ligament regeneration was observed by radiological means, HE staining and Sirus-red staining.Results Compared with collagen membrane group and blank control group, there was a clearly periodontal ligament like tissue and Sharpey′s like fibres formation in test group only.Conclustion Cell sheet-collagen membrane-cell sheet sandwich complex can effectively improve the periodontal ligament regeneration.
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Objective:To study the effects of endothelial progenitor cells(EPCs)on the osteogenesis of bone marrow mesenchymal stem cell (BMSCs)sheet-implant complex.Methods:EPCs were added to the BMSC sheets,and the expression of osteogenesis-relat-ed genes was examined by real time PCR.Cell sheets were wrapped around implants to construct cell sheet-implant complexes and the complexes were subcutaneously transplanted into SCID mice.The complexes were harvested 8 weeks after operation and observed by micro-CT and histological examination.Results:The BMSC sheet with EPCs showed higher expression of Runx2,ALP,BMP2 and VEGF in the in vitro test;higher bone volume ratio,greater amount of new bone tissue and higher expression of Runx2 and BMP2 in the in vivo test.Conclusion:EPCs can improve the osteogenesis of BMSC sheet-implant complex.
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The repair of bone defects poses a great challenge for reconstructive surgeons. Although the development of tissue engineering has exhibited promise in replacing damaged bone, the fabrication of large constructs with functional blood vessels remains an obstacle. From the orthopedic surgeon's point of view, the generation of axially vascularized bone, which can anastomose with the recipient vessel, might be a solution to this medical problem. In this study, we aimed to prefabricate an axially vascularized bone by combining a β-TCP scaffold, arteriovenous loop (AVL), and cell sheet in a bioreactor in vivo. Twelve rabbits were randomly allocated into two groups: the experimental group (presence of AVL) and the control group (absence of AVL). The constructs were explanted at 8 weeks postoperatively. The histomorphometric results showed 42.8±5.9% of the bone area in the AVL group and 26.6±3.5% in the control group. Similarly, vessel analysis revealed the average vessel density in the AVL group (12.5±3.3) was significantly more than that in the control group (6.1±1.5, p<0.05). Our research indicated that the combination of a β-TCP scaffold, AVL and cell sheet might engineer vascularized bone. This prefabrication strategy might facilitate clinical translation of bone tissue engineering in reconstructing large bone defects.
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Rabbits , Bioreactors , Blood Vessels , Bone and Bones , Orthopedics , Surgeons , Tissue EngineeringABSTRACT
Background Development of corneal tissue engineering creates a new therapeutic method for severe corneal diseases.However,ideal seed cells and scaffold for corneal surface reconstruction have not yet been investigated well.Adipose-derived stem cells (ADSCs) are varified to have a self-renewal ability and epithelioid features,and temperature-responsive scaffolds (TRSs) can offer technical support for stem cell sheet.Objective This study was to investigate the characteristics of ADSCs cultured on TRSs and compare these features to typical oral mucosal epithelial cells (OMECs),and therefore to explore the feasibility of reconstruction of ocular surface with ADSCs as seed cells.Methods Self-made TRSs were prepared by adding isopropyl alcohol dissolved poly-Nisopropylacrylamide (PNIPAAm) to each polystyrene tissue culture dish and then irradiating using an election beam.Subcutaneious fatty tissue of rabbit neck was obtained to culture ADSCs,and 4 pieces of oral cavity mucosal tissue were digested and cultured to obtain OMECs.Then the ADSCs and OMECs were incubated on TRSs,and cell morphology,growth rate,detached duration and survival counts were compared between ADSCs and OMECs.The ADSCs sheet and OMECs sheet were stained with hematoxylin and eosin for morphological examination.Immunochemistry was used to observe the expressions of stem-cell biomakers and epithelioid-cell biomakers in the cells.The ultrastructure of cell surface was observed under the scanning electron microscope.Results Self-made TRSs were similar to ordinary culture dish in the transparancy and smoothness.The water contact angle of 4 in 5 samples were ＞10° with the effective rate upto 80％.A DSCs showed the elongated fusiform in shape,while OMECs showed a cobblestone appearance.The growth cycle,detached duration and cell number of ADSCs were 12-14 days,(46.0 ±9.6) minutes and (7.9 ±1.1)×105/sheet,and those of OMECs were 14-16 days,(91.9 ±10.9) minutes and (45.8 ±26.5)×105/sheet,respectively,showing statistically significant differences in the detached duration and cell counts between ADSCs and OMECs (P=0.002,0.028).Hematoxylin and eosin staining showed that ADSCs sheet comprised only 1-3 layer cells,while OMECs showed 4-5 layer cells.ATP-binding cassette superfamily G member 2 (ABCG2),p63 and cytokeratin 12 (CK12) were positively expressed in both ADSCs sheet and OMECs sheet.Closely packed cells and typical eithelial microvilli in the cell surface were exhibited in both ADSCs sheet and OMECs sheet under the scanning electron microscope.Conclusions Self-made TRSs can be used as scaffold of ADSCs.The ADSCs sheet on the TRSs appears to have a good cell vitality and therefore is a new seed source of ocular surface reconstruction.
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As a novel concept for cell delivery,cell sheet may retain the extracellular matrix and adhesive proteins,avoid the use of bioma-terials for delivery,and increase cell survival rate while reduce cell loss following cell transplantation.This review summarizes the use of cell sheet technology for periodontal and pulp-dentin complex regeneration,highlights recent progresses and future challenges in this field.
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Objective:To observe the effects of dentin proseeded with BMSCs or PDLCs incorporated with PDLCs sheets and calci-nated ceramic bovine bone(CBB)in the reconstruction of periodontal tissue.Methods:Root dentin slices were prepared and pre-seeded with BMSCs or PDLCs from beagle dog.After cultivated in osteoinduction medium(OST groups)orα-modified eagle's medi-um(MEM groups)for 3 weeks,the dentin slices observed by scanning electron microscope (SEM).Then the slices were wrapped successively with PDLCs sheets and CBB as new constructs(BMSCs or PDLCs/dentin/PDLCs sheets/CBB).The new constructs were transplanted into nude mice subcutaneously,8 week after transplantation,samples were havested and examined by HE staining.Re-sults:Enough cells and extracellular matrices were detected on the dentin slices by SEM in vitro.A little new immature cementum-like tissue without periodontal-like tissue on the surface of the new construcs was observed in OST groups.By contrast,in MEM groups,periodontal-like without immature cementum-like tissue formation was observed.Conclusion:Dentin proseeded with BMSCs or PDLCs incorporated with PDLCs sheets and CBB cultured by MEM can promote periodontal-like tissue regeneration.Cultured with osteooinduction medium can promote cementum-like tissue regeneration.
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Objective To construct bone marrow stem cell sheets and to investigate its effects in the process of osteogenesis.Methods BMSCs were differentiated into osteoblasts and then seeded into a temperature responsive culture dish to construct BMSC sheets.PLGA scaffolds in which both BMSC suspension and BMSC sheets were added,were implanted into the left side of the dogs' mandible.In the other side,PLGA scaffolds that were not wapped with BMSC sheets were implanted as control.At 16 weeks,the samples were processed for radiological analysis and histological examination.Results Cells in the BMSC sheets grew well.In the experimental side,the optical density of the samples was higher than that of the control side (P＜0.05) and plenty of lamellar bones and Haversian system were observed.Conclusions The formation of lamellar bones can be promoted by PLGA scaffolds and BMSC sheets in the process of tissue engineering bone reconstrution.
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We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintaed intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.