Publications by authors named "Teruo Okano"

549 Publications

Strategies to address mesenchymal stem/stromal cell heterogeneity in immunomodulatory profiles to improve cell-based therapies.

Acta Biomater 2021 Apr 20. Epub 2021 Apr 20.

Cell Sheet Tissue Engineering Center (CSTEC), University of Utah, Salt Lake City, Utah, USA; Institute for Advanced Biomedical Sciences, Tokyo Women's Medical University, Tokyo, Japan. Electronic address:

Mesenchymal stromal cells (MSCs) have gained immense attention over the past two decades due to their multipotent differentiation potential and pro-regenerative and immunomodulatory cytokine secretory profiles. Their ability to modulate the host immune system and promote tolerance has prompted several allogeneic and autologous hMSC-based clinical trials for the treatment of graft-versus-host disease and several other immune-induced disorders. However, clinical success beyond safety is still controversial and highly variable, with inconclusive therapeutic benefits and little mechanistic explanation. This clinical variability has been broadly attributed to inconsistent MSC sourcing, phenotypic characterization, variable potency, and non-standard isolation protocols, leading to functional heterogeneity among administered MSCs. Homogeneous MSC populations are proposed to yield more predictable, reliable biological responses and clinically meaningful properties relevant to cell-based therapies. Limited comparisons of heterogeneous MSCs with homogenous MSCs are reported. This review addresses this gap in the literature with a critical analysis of strategies aimed at decreasing MSC heterogeneity concerning their reported immunomodulatory profiles. STATEMENT OF SIGNIFICANCE: This review collates, summarizes, and critically analyzes published strategies that seek to improve homogeneity in immunomodulatory functioning MSC populations intended as cell therapies to treat immune-based disorders, such as graft-vs-host-disease. No such review for MSC therapies, immunomodulatory profiles and cell heterogeneity analysis is published. Since MSCs represent the most clinically studied experimental cell therapy platform globally for which there remains no US domestic marketing approval, insights into MSC challenges in therapeutic product development are imperative to providing solutions for immunomodulatory variabilities.
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http://dx.doi.org/10.1016/j.actbio.2021.03.069DOI Listing
April 2021

3D cell sheet structure augments mesenchymal stem cell cytokine production.

Sci Rep 2021 Apr 14;11(1):8170. Epub 2021 Apr 14.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, 30 South 2000 East, Salt Lake City, UT, 84112, USA.

Mesenchymal stem cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. An increasing body of evidence suggests that this paracrine function is enhanced by MSC cultivation in three-dimensional (3D) tissue-like microenvironments. Toward this end, this study explored scaffold-free cell sheet technology as a new 3D platform. MSCs cultivated on temperature-responsive culture dishes to a confluent 2D monolayer were harvested by temperature reduction from 37 to 20 °C that induces a surface wettability transition from hydrophobic to hydrophilic. Release of culture-adherent tension induced spontaneous cell sheet contraction, reducing the diameter 2.4-fold, and increasing the thickness 8.0-fold to render a 3D tissue-like construct with a 36% increase in tissue volume. This 2D-to-3D transition reorganized MSC actin cytoskeleton from aligned to multidirectional, corresponding to a cell morphological change from elongated in 2D monolayers to rounded in 3D cell sheets. 3D culture increased MSC gene expression of cell interaction proteins, β-catenin, integrin β1, and connexin 43, and of pro-tissue regenerative cytokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10), and increased VEGF secretion per MSC 2.1-fold relative to 2D cultures. Together, these findings demonstrate that MSC therapeutic potency can be enhanced by 3D cell sheet tissue structure.
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http://dx.doi.org/10.1038/s41598-021-87571-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046983PMC
April 2021

Trends in Articular Cartilage Tissue Engineering: 3D Mesenchymal Stem Cell Sheets as Candidates for Engineered Hyaline-Like Cartilage.

Cells 2021 Mar 13;10(3). Epub 2021 Mar 13.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 South 2000 East, Salt Lake City, UT 84112, USA.

Articular cartilage defects represent an inciting factor for future osteoarthritis (OA) and degenerative joint disease progression. Despite multiple clinically available therapies that succeed in providing short term pain reduction and restoration of limited mobility, current treatments do not reliably regenerate native hyaline cartilage or halt cartilage degeneration at these defect sites. Novel therapeutics aimed at addressing limitations of current clinical cartilage regeneration therapies increasingly focus on allogeneic cells, specifically mesenchymal stem cells (MSCs), as potent, banked, and available cell sources that express chondrogenic lineage commitment capabilities. Innovative tissue engineering approaches employing allogeneic MSCs aim to develop three-dimensional (3D), chondrogenically differentiated constructs for direct and immediate replacement of hyaline cartilage, improve local site tissue integration, and optimize treatment outcomes. Among emerging tissue engineering technologies, advancements in cell sheet tissue engineering offer promising capabilities for achieving both in vitro hyaline-like differentiation and effective transplantation, based on controlled 3D cellular interactions and retained cellular adhesion molecules. This review focuses on 3D MSC-based tissue engineering approaches for fabricating "ready-to-use" hyaline-like cartilage constructs for future rapid in vivo regenerative cartilage therapies. We highlight current approaches and future directions regarding development of MSC-derived cartilage therapies, emphasizing cell sheet tissue engineering, with specific focus on regulating 3D cellular interactions for controlled chondrogenic differentiation and post-differentiation transplantation capabilities.
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http://dx.doi.org/10.3390/cells10030643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998529PMC
March 2021

Improvement of the therapeutic capacity of insulin-producing cells trans-differentiated from human liver cells using engineered cell sheet.

Stem Cell Res Ther 2021 Jan 6;12(1). Epub 2021 Jan 6.

Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.

Background: Although pancreatic islet transplantation therapy is ideal for diabetes patients, several hurdles have prevented it from becoming a standard treatment, including donor shortage and low engraftment efficacy. In this study, we prepared insulin-producing cells trans-differentiated from adult human liver cells as a new islet source. Also, cell sheet formation could improve differentiation efficiency and graft survival.

Methods: Liver cells were expanded in vitro and trans-differentiated to IPCs using adenovirus vectors carrying human genes for PDX1, NEUROD1, and MAFA. IPCs were seeded on temperature-responsive culture dishes to form cell sheets. Differentiation efficiency was confirmed by ß cell-specific gene expression, insulin production, and immunohistochemistry. IPC suspension was injected by portal vein (PV), and IPC sheet was transplanted on the liver surface of the diabetic nude mouse. The therapeutic effect of IPC sheet was evaluated by comparing blood glucose control, weight gain, histological evaluation, and hepatotoxicity with IPC injection group. Also, cell biodistribution was assessed by in vivo/ex vivo fluorescence image tagging.

Results: Insulin gene expression and protein production were significantly increased on IPC sheets compared with those in IPCs cultured on conventional culture dishes. Transplanted IPC sheets displayed significantly higher engraftment efficiency and fewer transplanted cells in other organs than injected IPCs, and also lower liver toxicity, improved blood glucose levels, and weight gain. Immunohistochemical analyses of liver tissue revealed positive staining for PDX1 and insulin at 1, 2, and 4 weeks after IPC transplantation.

Conclusions: In conclusion, cell sheet formation enhanced the differentiation function and maturation of IPCs in vitro. Additionally, parameters for clinical application such as distribution, therapeutic efficacy, and toxicity were favorable. The cell sheet technique may be used with IPCs derived from various cell sources in clinical applications.
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http://dx.doi.org/10.1186/s13287-020-02080-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786992PMC
January 2021

Capillary Networks for Bio-Artificial Three-Dimensional Tissues Fabricated Using Cell Sheet Based Tissue Engineering.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Center for Advanced Medical and Life Science, Tokyo Women's Medical University, Tokyo 162-8666, Japan.

One of the most important challenges facing researchers in the field of regenerative medicine is to develop methods to introduce vascular networks into bioengineered tissues. Although cell scaffolds that slowly release angiogenic factors can promote post-transplantation angiogenesis, they cannot be used to construct thick tissues because of the time required for sufficient vascular network formation. Recently, the co-culture of graft tissue with vascular cells before transplantation has attracted attention as a way of promoting capillary angiogenesis. Although the co-cultured vascular cells can directly contribute to blood vessel formation within the tissue, a key objective that needs to be met is the construction of a continuous circulatory structure. Previously described strategies to reconstruct blood vessels include the culture of endothelial cells in a scaffold that contains microchannels or within the original vascular framework after decellularization of an entire organ. The technique, as developed by authors, involves the progressive stacking of three-layered cell sheets onto a vascular bed to induce the formation of a capillary network within the cell sheets. This approach enables the construction of thick, functional tissue of high cell density that can be transplanted by anastomosing its artery and vein (provided by the vascular bed) with host blood vessels.
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http://dx.doi.org/10.3390/ijms22010092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795136PMC
December 2020

Micropatterned Smart Culture Surfaces via Multi-Step Physical Coating of Functional Block Copolymers for Harvesting Cell Sheets with Controlled Sizes and Shapes.

Macromol Biosci 2021 03 28;21(3):e2000330. Epub 2020 Dec 28.

Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku, Tokyo, 162-8666, Japan.

Cell micropatterning on micropatterned thermoresponsive polymer-based culture surfaces facilitates the creation of on-demand and functional cell sheets. However, the fabrication of micropatterned surfaces generally includes complicated procedures with multi-step chemical reactions. To overcome this issue, this study proposes a facile preparation of micropatterned thermoresponsive surfaces via a two-step physical coating of two different diblock copolymers. Both copolymers contain poly(butyl methacrylate) blocks as hydrophobic anchors for water-stable polymer deposition. At first, thermoresponsive polymer layers are constructed on cell culture dishes via spin-coating block copolymers containing poly(N-isopropylacrylamide) blocks that exhibit a transition temperature of ≈30 °C in aqueous media. To create polymer micropatterns on the thermoresponsive surfaces, microcontact printing of block copolymers containing hydrophilic poly(N-acryloylmorpholine) (PNAM) blocks is performed using polydimethylsiloxane stamps. Stamped PNAM-based block polymers are adsorbed to the outermost thermoresponsive surfaces, and increase the surface hydrophilicity with decreasing protein adsorption. Cells adhere and proliferate on the thermoresponsive domains at 37 °C, whereas the stamped hydrophilic domains remain cell-repellent for 7 days. At 20 °C, cell sheets with controlled sizes and shapes are harvested from the surfaces with the desired micropatterns. This technique is useful for the preparation of micropatterned polymer surfaces for various biomedical applications.
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http://dx.doi.org/10.1002/mabi.202000330DOI Listing
March 2021

Human Mesenchymal Stem Cell Sheets Improve Uterine Incision Repair in a Rodent Hysterotomy Model.

Am J Perinatol 2020 Dec 27. Epub 2020 Dec 27.

Cell Sheet Tissue Engineering Center, Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, Salt Lake City, Utah.

Objective:  The study aimed to assess the feasibility of creating and transplanting human umbilical cord mesenchymal stem cell sheets applied to a rat model of hysterotomy, and additionally to determine benefits of human umbilical cord mesenchymal stem cell sheet transplantation in reducing uterine fibrosis and scarring.

Study Design:  Human umbilical cord mesenchymal stem cell sheets are generated by culturing human umbilical cord mesenchymal stem cells on thermo-responsive cell culture plates. The temperature-sensitive property of these culture dishes facilitates normal cell culture in a thin contiguous layer and allows for reliable recovery of intact stem cell sheets without use of destructive proteolytic enzymes.We developed a rat hysterotomy model using nude rats. The rat uterus has two distinct horns: one horn provided a control/untreated scarring site, while the second horn was the cell sheet transplantation site.On day 14 following surgery, complete uteri were harvested and subjected to histologic evaluations of all hysterotomy sites.

Results:  The stem cell sheet culture process yielded human umbilical cord mesenchymal stem cell sheets with surface area of approximately 1 cm.Mean myometrial thickness in the cell sheet-transplanted group was 274 µm compared with 191 µm in the control group ( = 0.02). Mean fibrotic surface area in the human umbilical cord mesenchymal stem cell sheet-transplanted group was 95,861 µm compared with 129,185 µm in the control group. Compared with control horn sites, cell sheet-transplanted horns exhibited significantly smaller fibrotic-to-normal myometrium ratios (0.18 vs. 0.27, respectively,  = 0.029). Mean number of fibroblasts in cell sheet-transplanted horns was significantly smaller than the control horns (483 vs. 716/mm, respectively,  = 0.001).

Conclusion:  Human umbilical cord mesenchymal stem cell sheet transplantation is feasible in a rat model of hysterotomy. Furthermore, use of stem cell sheets reduces fibroblast infiltration and uterine scar fibrotic tissue formation during hysterotomy healing, potentially mitigating risks of uterine scar formation.

Key Points: · Stem cell sheet transplanted to hysterotomy promotes myometrial regeneration and reduced fibrotic tissue formation.. · This study demonstrates the feasibility of using human umbilical cord mesenchymal stem cell sheets..
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http://dx.doi.org/10.1055/s-0040-1721718DOI Listing
December 2020

Allogeneic mesenchymal stem cell sheet therapy: A new frontier in drug delivery systems.

J Control Release 2021 Feb 19;330:696-704. Epub 2020 Dec 19.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, 30 South 2000 East, Salt Lake City, UT 84112, USA; Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

The evolution of drug discovery exploded in the early 20th century with the advent of critical scientific advancements in organic chemistry, chemical analysis, and purification. Early drug generations focused largely on symptom control and pain management, effective targets for small-molecule drugs. Recently, the attention in drug discovery has shifted to pursuit of radical cures. Cell therapy presents the ideal attributes of a promising new drug, targeting specific tissues based on chemotactic cues and modulating secretion of instructive regenerative molecules in response to dynamic signaling from disease environments. To actuate the therapeutic potential of cell therapy toward worldwide clinical use, cell delivery methods that can effectively localize and engraft mesenchymal stem cells (MSCs) with high disease-site fidelity and enable dynamic MSC bioactive function are paramount. In this review, we discuss the evolution of cell therapies with a focus on stem cell advantages, as well as the limitations to these therapies. This review aims to introduce cell sheet technology as a breakthrough cell therapy with demonstrated therapeutic success across indications for heart, liver, and kidney tissue regeneration. Opportunities and anticipated clinical impacts of cell sheet technology using MSCs are discussed.
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http://dx.doi.org/10.1016/j.jconrel.2020.12.028DOI Listing
February 2021

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands.

Cells 2020 12 9;9(12). Epub 2020 Dec 9.

Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.
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http://dx.doi.org/10.3390/cells9122645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763220PMC
December 2020

Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets.

Sci Rep 2020 11 30;10(1):20869. Epub 2020 Nov 30.

Department of Pharmaceutics and Pharmaceutical Chemistry, Cell Sheet Tissue Engineering Center (CSTEC), University of Utah, 30 South 2000 East, Salt Lake City, UT, 84112, USA.

Cell and tissue engineering approaches for articular cartilage regeneration increasingly focus on mesenchymal stem cells (MSCs) as allogeneic cell sources, based on availability and innate chondrogenic potential. Many MSCs exhibit chondrogenic potential as three-dimensional (3D) cultures (i.e. pellets and seeded biomaterial scaffolds) in vitro; however, these constructs present engraftment, biocompatibility, and cell functionality limitations in vivo. Cell sheet technology maintains cell functionality as scaffold-free constructs while enabling direct cell transplantation from in vitro culture to targeted sites in vivo. The present study aims to develop transplantable hyaline-like cartilage constructs by stimulating MSC chondrogenic differentiation as cell sheets. To achieve this goal, 3D MSC sheets are prepared, exploiting spontaneous post-detachment cell sheet contraction, and chondrogenically induced. Results support 3D MSC sheets' chondrogenic differentiation to hyaline cartilage in vitro via post-contraction cytoskeletal reorganization and structural transformations. These 3D cell sheets' initial thickness and cellular densities may also modulate MSC-derived chondrocyte hypertrophy in vitro. Furthermore, chondrogenically differentiated cell sheets adhere directly to cartilage surfaces via retention of adhesion molecules while maintaining the cell sheets' characteristics. Together, these data support the utility of cell sheet technology for fabricating scaffold-free, hyaline-like cartilage constructs from MSCs for future transplantable articular cartilage regeneration therapies.
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http://dx.doi.org/10.1038/s41598-020-77842-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705723PMC
November 2020

Novel therapies using cell sheets engineered from allogeneic mesenchymal stem/stromal cells.

Emerg Top Life Sci 2020 12;4(6):677-689

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, 30 South 2000 East, Salt Lake City, Utah 84112, U.S.A.

Mesenchymal stem/stromal cells (MSCs) have long been recognized to help regenerate tissues, by exploiting their intrinsic potentials for differentiation and secretion of therapeutic paracrine factors together with feasibility for cell banking. These unique MSC properties are attractive to provide effective new cell-based therapies for unmet medical needs. Currently, the infusion of suspended MSCs is accepted as a promising therapy to treat systemic inflammatory diseases. However, low cell engraftment/retention in target organs and off-target entrapment using conventional cell infusion must be improved to provide reliable localized disease treatments. Cell sheet technology offers an alternative: three-dimensional (3D) tissue-like structures can be harvested from culture using mild temperature reduction, and transplanted directly onto target tissue sites without suturing, yielding stable cell engraftment and prolonged cell retention in situ without off-target losses. Engineered MSC sheets directly address two major cell therapy strategies based on their therapeutic benefits: (1) tissue replacements based on mult-ilineage differentiation capacities, focusing on cartilage regeneration in this review, and (2) enhancement of tissue recovery via paracrine signaling, employing their various secreted cytokines to promote neovascularization. MSCs also have production benefits as a promising allogeneic cell source by exploiting their reliable proliferative capacity to facilitate expansion and sustainable cell banking for off-the-shelf therapies. This article reviews the advantages of both MSCs as allogeneic cell sources in contrast with autologous cell sources, and allogeneic MSC sheets engineered on thermo-responsive cell dishes as determined in basic studies and clinical achievements, indicating promise to provide robust new cell therapies to future patients.
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http://dx.doi.org/10.1042/ETLS20200151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939697PMC
December 2020

Femoral Head Chondrocyte Viability at the Cam Deformity in Patients With Femoroacetabular Impingement Syndrome.

Am J Sports Med 2020 12 27;48(14):3586-3593. Epub 2020 Oct 27.

Department of Orthopaedic Surgery, University of Utah, Salt Lake City, Utah, USA.

Background: Patients with hip pathology, such as femoroacetabular impingement (FAI) or hip dysplasia, are known to sustain chondral delamination injuries identifiable during hip arthroscopy, with an incidence of 44% to 75%. There are studies focused on understanding acetabular chondral flap viability, but there is a dearth of research regarding the viability of femoral head cartilage overlying the cam deformity in FAI.

Purpose: To describe the viability and immunohistochemistry staining patterns of femoral head cartilage in the setting of FAI.

Study Design: Descriptive laboratory study.

Methods: Between September 2018 and August 2019, a single surgeon prospectively collected full-thickness femoral cartilage from cam deformities in 14 patients with FAI undergoing osteoplasty. Samples were assessed for viability and underwent immunohistochemistry staining for collagen type I, collagen type II, and aggrecan.

Results: The data set included 14 patients. Twelve samples were assessed for viability and 14 for immunohistochemistry straining. The mean patient age was 34.1 years, and the mean body mass index was 24.69. Mean ± SD chondrocyte viability per patient was 52% ± 11%. At the time of cell isolation, 8 of the 12 patients had viability >50%, with a maximum of 68.2%. This viability increased after a primary culture period, varying from 9 to 13 days, with 10 of 12 samples having viability >90%. The viability mean after the culture period was 94.54% ± 4.89%. Harvested cartilage showed expressions of type I cartilage, type II collagen, and aggrecan in a pattern that is predictable for native cartilage.

Conclusion: These data reveal that the cartilage in femoral head cartilage overlying cam deformity-much like that from acetabular chondral flaps-not only has baseline viability >50% (51.99% ± 10.83%) but the ability to increase in viability >90% after a culture period. There may be a role for use of femoral head cartilage as autograft to repair full-thickness cartilage defects of the acetabulum and femoral head, either at the time of osteochondroplasty or after a period of cell culture to improve cell viability.

Clinical Relevance: A dearth of information is available regarding the viability of femoral head cartilage. This study provides insight into the cartilage viability and response to culture.
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http://dx.doi.org/10.1177/0363546520962788DOI Listing
December 2020

Evaluation of Multi-Layered Pancreatic Islets and Adipose-Derived Stem Cell Sheets Transplanted on Various Sites for Diabetes Treatment.

Cells 2020 08 31;9(9). Epub 2020 Aug 31.

Department of Medical Science, Asan Medical Institute of Convergence Science and Technology (AMIST), Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea.

Islet cell transplantation is considered an ideal treatment for insulin-deficient diabetes, but implantation sites are limited and show low graft survival. Cell sheet technology and adipose-derived stem cells (ADSCs) can be useful tools for improving islet cell transplantation outcomes since both can increase implantation efficacy and graft survival. Herein, the optimal transplantation site in diabetic mice was investigated using islets and stem cell sheets. We constructed multi-layered cell sheets using rat/human islets and human ADSCs. Cell sheets were fabricated using temperature-responsive culture dishes. Islet/ADSC sheet (AI sheet) group showed higher viability and glucose-stimulated insulin secretion than islet-only group. Compared to islet transplantation alone, subcutaneous AI sheet transplantation showed better blood glucose control and CD31+ vascular traits. Because of the adhesive properties of cell sheets, AI sheets were easily applied on liver and peritoneal surfaces. Liver or peritoneal surface grafts showed better glucose control, weight gain, and intraperitoneal glucose tolerance test (IPGTT) profiles than subcutaneous site grafts using both rat and human islets. Stem cell sheets increased the therapeutic efficacy of islets in vivo because mesenchymal stem cells enhance islet function and induce neovascularization around transplanted islets. The liver and peritoneal surface can be used more effectively than the subcutaneous site in future clinical applications.
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http://dx.doi.org/10.3390/cells9091999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563383PMC
August 2020

Water stable nanocoatings of poly(N-isopropylacrylamide)-based block copolymers on culture insert membranes for temperature-controlled cell adhesion.

J Mater Chem B 2020 09 4;8(34):7812-7821. Epub 2020 Aug 4.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan.

This study demonstrated the spin-coating of functional diblock copolymers to develop smart culture inserts for thermoresponsive cell adhesion/detachment control. One part of the block components, the poly(n-butyl methacrylate) block, strongly supported the water stable surface-immobilization of the thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) block, regardless of temperature. The chain length of the PNIPAAm blocks was varied to regulate thermal surface functions. Immobilized PNIPAAm concentrations became larger with increasing chain length (1.0-1.6 μg cm) and the thicknesses of individual layers were relatively comparable at 10-odd nanometers. A nanothin coating scarcely inhibited the permeability of the original porous membrane. When human fibroblasts were cultured on each surface at 37 °C, the efficiencies of cell adhesion and proliferation decreased with longer PNIPAAm chains. Meanwhile, by reducing the temperature to 20 °C, longer PNIPAAm chains promoted cell detachment owing to the significant thermoresponsive alteration of cell-surface affinity. Consequently, we successfully produced a favorable cell sheet by choosing an appropriate PNIPAAm length for block copolymers.
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http://dx.doi.org/10.1039/d0tb01113dDOI Listing
September 2020

Enhanced mechanical properties and cell separation with thermal control of PIPAAm-brushed polymer-blend microfibers.

J Mater Chem B 2020 07 23;8(28):6017-6026. Epub 2020 Jun 23.

Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan.

We have developed thermoresponsive microfibers with improved mechanical properties and enhanced temperature modulated-cell separation. Microfiber substrates were electrospun using poly(4-vinylbenzyl chloride) (PVBC)-poly(n-butyl methacrylate) (PBMA) blend materials in different ratios. Although their diameters were similar to those of the PVBC homofibers, polymer-blend microfibers exhibited excellent mechanical properties including non-brittle softness, owing to PBMA with a low T. These polymer-blend microfibers enabled the preparation of thin, dense mats that were superior in the experimental handling of cell separation. Poly(N-isopropylacrylamide) (PIPAAm) brushes were grafted via surface-initiated atom transfer radical polymerization from the initiation sites of PVBC in the polymer-blend microfiber substrates. The microfiber in a 25 : 75 ratio of PVBC : PBMA had a reasonable amount of the initiation sites and superior mechanical properties. The PIPAAm-brushed microfibers of the 25 : 75 blend substrate were capable of temperature-modulation, both in terms of wettability and cell separation. Among the normal human dermal fibroblasts (NHDFs), human umbilical vein endothelial cells (HUVECs), and human skeletal muscle myoblasts (HSMMs), HUVEC cells showed significantly poor adhesion on fibers at 37 °C; they were separated from adhered NHDF and HSMM cells in the initial step. Reducing the temperature to 20 °C remarkably detached NHDF cells, allowing their separation from HSMM cells. Compared with the PIPAAm-brushed PVBC homopolymer microfibers, these cell-separating functions were enhanced in the thermoresponsive PBMA-rich polymer-blend microfibers, probably ascribed to the properties of PBMA and the moderate density of the PIPAAm-brush. Thus, the developed microfibers could be useful for temperature-modulated cell separation systems.
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http://dx.doi.org/10.1039/d0tb00972eDOI Listing
July 2020

Professor Sung Wan Kim - A pioneer in the world of pharmaceutics; biomedical polymer.

Regen Ther 2020 Jun 22;14:330-331. Epub 2020 May 22.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, 30 South 2000 East, Salt Lake City, UT 84112, USA.

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http://dx.doi.org/10.1016/j.reth.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248284PMC
June 2020

Cell sheet tissue engineering for scaffold-free three-dimensional (3D) tissue reconstruction.

Methods Cell Biol 2020 21;157:143-167. Epub 2020 Jan 21.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, Salt Lake City, UT, United States; Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan. Electronic address:

Three-dimensional (3D) reconstruction of highly functional tissues is of great importance in advancing the clinical benefit of tissue engineering and regenerative medicine. In the last quarter century, many studies have found that by engineering a 3D microenvironment that resembles the in vivo tissue condition, cells exhibit behaviors and functions that reflect those of native tissue. Biomaterial scaffolds are a central technology for providing 3D microenvironments in vitro, and, in conjunction with diverse design and cell seeding advents, have produced highly functional and complex 3D tissues. Here, we describe a new approach to creating 3D cell-dense tissue-like constructs without a biomaterial scaffold. Cell sheet technology with cell sheet layering strategies generates highly cell dense, engineered tissue capable of direct crosstalk with the tissue-engraftment surface, in addition to paracrine-mediated signaling. In this chapter, we will introduce methods of reconstructing 3D tissue using cell sheet technology and the advantages of a scaffold-free design.
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http://dx.doi.org/10.1016/bs.mcb.2019.11.020DOI Listing
January 2020

Stable cell adhesion affects mesenchymal stem cell sheet fabrication: Effects of fetal bovine serum and human platelet lysate.

J Tissue Eng Regen Med 2020 05 6;14(5):741-753. Epub 2020 Apr 6.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, Salt Lake City, UT, USA.

Cell sheet technology exploits temperature responsive cell culture dishes (TRCDs) as versatile cell harvesting methods to yield contiguous cell monolayers robustly held together by cell-cell junctions, receptors, and endogenous extracellular matrix. More than 15 years of clinical data using autologous-sourced cell sheets demonstrate enhanced therapeutic properties through increased cell retention at target tissue sites. Recently, several preclinical studies have also been reported using mesenchymal stem cell (MSC) sheets in wound healing, cardiac ischemia therapies, and pancreatic regeneration. However, optimized MSC sheet fabrication conditions have not yet been reported. In this study, we identified specific conditions for reliable human MSC sheet fabrication by comparing cell growth media supplements (fetal bovine serum [FBS] and human platelet lysate [hPL]). Human umbilical cord-derived MSCs cultured in FBS and hPL exhibit different actin cytoskeletal structures related to their cell morphologies and adhesion. MSCs cultured in FBS media showed stable cell adhesion on TRCDs with flattened cell shapes and aligned actin cytoskeletal structure. This stable cell adhesion enables production of consistent MSC cell sheets, with controlled cell sheet detachment. Conversely, cell sheet fabrication in hPL media exhibits poor reproducibility being more sensitive to temperature- and culture time-induced release due to weak cell adhesion. These findings suggest that stable MSC adhesion to TRCDs is important to reliable MSC sheet fabrication methods and that MSC growth media supplementation directly affects cell adhesion during culture.
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http://dx.doi.org/10.1002/term.3037DOI Listing
May 2020

A stable protocol for the fabrication of transplantable human oral mucosal epithelial cell sheets for clinical application.

Regen Ther 2020 Jun 16;14:87-94. Epub 2020 Jan 16.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-Cho, Shinjuku-ku, Tokyo, 162-8666, Japan.

Introduction: Cultured stratified epithelial cell sheets have been clinically utilized as transplantable grafts for the regeneration of epithelial tissues, such as the esophagus, cornea, skin, and intraoral cavity. These cell sheets are expected to gain widespread use as regenerative medicine products and save many patients. For this purpose, establishing and disseminating the stale protocol of fabricating the cell sheet is crucial. The fabrication of cultured stratified epithelial cell sheets consists of many important steps, and since the patients' epithelial cell conditions vary widely and are sometimes unstable, the qualities of the epithelial cell grafts are likewise potentially unstable. Therefore, in this paper, we report the stable protocol for fabrication of the transplantable cell sheet particularly from patient-derived oral mucosal tissues.

Methods: Serum extracted from blood and buccal mucosal tissue were collected in Nagasaki University and transported to Tokyo Women's Medical University. Oral mucosal epithelial cells were collected by minimum trypsin method, and this treatment was studied whether to be a critical procedure. After 14 days cultivation, cultured cells were examined whether to be transplantable as cell sheets.

Results: We successfully transported buccal mucosal tissue and serum without damage and contamination. Oral mucosal epithelial cells were collected with high viability by minimum trypsin method. Finally, we succeeded to stably fabricate oral mucosal epithelial cell sheets in all 10 patients.

Conclusions: We established a stable protocol for the fabrication of human oral mucosal epithelial cell sheets and their transportation in clinical settings in this study. These methodologies could also be basis for transplantation therapy using cultured cell sheets of various types other than oral mucosal epithelial cell and will contribute largely to the future development of regenerative medicine.
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http://dx.doi.org/10.1016/j.reth.2019.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970131PMC
June 2020

Microfluidic vascular-bed devices for vascularized 3D tissue engineering: tissue engineering on a chip.

Biomed Microdevices 2019 12 20;22(1). Epub 2019 Dec 20.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-Cho, Shinjuku-Ku, Tokyo, 162-8666, Japan.

In this report, we describe a microfluidic vascular-bed (micro-VB) device providing a platform for 3D tissue engineering with vascular network formation. The micro-VB device allows functional connections between endothelial capillaries of heterogeneous sections (5-100 μm in diameter) and artificial plastic tubes or reservoirs (1-10 mm in diameter). Moreover, the micro-VB device can be installed in a standard 100 mm-diameter Petri dish. Endothelial networks in 3D engineered tissues were obtained by cellular self-assembly on the device, after co-culturing of human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) in fibrin gel. Endothelial capillary connection between vascularized tissues and microfluidic channels, mimicking arteries and veins, was confirmed by perfusion of fluorescent microspheres. The micro-VB devices were compatible with the use of commercially available culture dishes and did not require the involvement of additional equipment. Thus, these micro-VB devices are expected to substantially improve the routine application of 3D tissue engineering to regenerative medicine.
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http://dx.doi.org/10.1007/s10544-019-0461-2DOI Listing
December 2019

Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets.

Stem Cell Res Ther 2019 11 28;10(1):353. Epub 2019 Nov 28.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Health Sciences, 30 South 2000 East, Salt Lake City, UT, 84112, USA.

Background: In most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC therapeutic efficacies are inconsistent, largely attributed to transplanted MSC difficulties both in engrafting at tissue sites and in retaining their therapeutic functions from suspension formulations. MSC functional components, including cell adhesion and cell-cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Cell sheet technology, exploiting temperature-responsive cell culture surfaces, permits cell harvest without cell protein damage. This study is focused on phenotypic traits of MSC sheets structurally and functionally to understand therapeutic benefits of cell sheets.

Methods/results: This study verified cleaved cellular proteins (vinculin, fibronectin, laminin, integrin β-1, and connexin 43) and increased apoptotic cell death produced under standard trypsin harvesting treatment in a time-dependent manner. However, MSC sheets produced without trypsin using only temperature-controlled sheet harvest from culture plastic exhibited intact cellular structures. Also, MSCs harvested using enzymatic treatment (i.e., chemical disruption) showed higher pYAP expression compared to MSC sheets.

Conclusion: Retention of cellular structures such as ECM, cell-cell junctions, and cell-ECM junctions is correlated with human umbilical cord mesenchymal stem cell (hUC-MSC) survival after detachment from cell culture surfaces. Retaining these proteins intact in MSC cultures using cell sheet technology is proposed to enhance stem cell survival and their function in stem cell-based therapy.
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http://dx.doi.org/10.1186/s13287-019-1431-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883536PMC
November 2019

Fabrication of tissue-engineered cell sheets by automated cell culture equipment.

J Tissue Eng Regen Med 2019 12 14;13(12):2246-2255. Epub 2019 Nov 14.

Research & Development Group|, Hitachi, Ltd., Hatoyama, Japan.

Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.
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http://dx.doi.org/10.1002/term.2968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972683PMC
December 2019

Human mesenchymal stem cell sheets in xeno-free media for possible allogenic applications.

Sci Rep 2019 10 8;9(1):14415. Epub 2019 Oct 8.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, 30 South 2000 East, Salt Lake City, Utah, 84112, USA.

Cell-based therapies are increasingly focused on allogeneic stem cell sources because of several advantages in eliminating donor variability (e.g., aging and disease pathophysiology) affecting stem cell quality and in cell-banked sourcing of healthy donors to enable "off-the-shelf" products. However, allogeneic cell therapy is limited by host patient immunologic competence and inconsistent performance due to cell delivery methods. To address allogeneic cell therapy limitations, this study developed a new allogeneic stem cell sheet using human umbilical cord mesenchymal stem cells (hUC-MSC) that present low antigenicity (i.e., major histocompatibility complex, MHC). Optimal conditions including cell density, passage number, and culture time were examined to fabricate reliable hUC-MSC sheets. MHC II antigens correlated to alloimmune rejection were barely expressed in hUC-MSC sheets compared to other comparator MSC sheets (hBMSC and hADSC). hUC-MSC sheets easily graft spontaneously onto subcutaneous tissue in immune-deficient mice within 10 minutes of placement. No sutures are required to secure sheets to tissue because sheet extracellular matrix (ECM) actively facilitates cell-target tissue adhesion. At 10 days post-transplantation, hUC-MSC sheets remain on ectopic target tissue sites and exhibit new blood vessel formation. Furthermore, implanted hUC-MSC sheets secrete human HGF continuously to the murine target tissue. hUC-MSC sheets described here should provide new insights for improving allogenic cell-based therapies.
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http://dx.doi.org/10.1038/s41598-019-50430-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783458PMC
October 2019

Stable and Prolonged Autonomous Oscillation in a Self-Oscillating Polymer Brush Prepared on a Porous Glass Substrate.

Langmuir 2019 Jul 19;35(30):9794-9801. Epub 2019 Jul 19.

Department of Materials Engineering, School of Engineering , The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku , Tokyo 113-8656 , Japan.

We developed an autonomous functional surface, named a "self-oscillating polymer brush surface", which exhibits swelling-deswelling of the modified polymer chains synchronized with the Belousov-Zhabotinsky (BZ) reaction. The grafted polymer chain is a random copolymer composed of thermoresponsive -isopropylacrylamide, -(3-aminopropyl)methacrylamide, and ruthenium tris(2,2'-bipyridine) [Ru(bpy)]. To provide stable oscillations over a long period of time, suppression of the dilution of the BZ reactants inside the polymer surface and the increase in the amount of immobilized Ru(bpy) are important. Here, we modified the self-oscillating polymer brush on a porous glass substrate and characterized its dynamic behavior. The increased surface area of the porous glass allowed for an efficient introduction of the metal catalyst, which resulted in a stable BZ reaction observable by optical microscopy. Compared with an aqueous BZ solution and the self-oscillating polymer modified on a glass coverslip, the wave velocity and diffusion coefficient were significantly lower for the porous glass system, which suggested that the reaction-diffusion of the reactants was markedly different than those of the other two systems. Moreover, the wave velocity was unchanged on the porous glass system for 1 h, whereas that of the solution dropped by 30 μm s. Waveform analyses based on the Field-Körös-Noyes mechanism revealed that densely packed Ru(bpy) in the porous glass system affects the duration of the key processes in the BZ reaction. These findings can help with understanding the dynamic behavior of the self-oscillating polymer brush on a porous glass substrate. Stable self-oscillations on the polymer brush-grafted porous glass substrate will aid future applications such as mass transport systems.
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http://dx.doi.org/10.1021/acs.langmuir.9b00928DOI Listing
July 2019

Using cell sheets to regenerate mouse submandibular glands.

NPJ Regen Med 2019 4;4:16. Epub 2019 Jul 4.

1School of Dentistry, The University of Utah, Salt Lake City, UT USA.

Temperature-responsive polymer grafted tissue culture dishes release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining cell-cell communications, functional extracellular matrices and tissue-like behaviors. These features promote tissue regeneration and improve transplantation efficacy in various tissues including cartilage, heart, kidney, liver, endometrium, cornea, middle ear, periodontium, and esophageal living sheet transplants. However, the functional effects of cell sheets for salivary gland regeneration to treat hyposalivation have not yet been studied. Thus, the present study aims to both establish the viability of thermoresponsive cell sheets for use in salivary glands and then explore the delivery option (i.e., single vs. multiple layers) that would result in the most complete tissue growth in terms of cell differentiation and recovered tissue integrity. Results indicate that single cell sheets form polarized structures that maintain cell-cell junctions and secretory granules in vitro while layering of two-single cell sheets forms a glandular-like pattern in vitro. Moreover, double layer cell sheets enhance tissue formation, cell differentiation and saliva secretion in vivo. In contrast, single cell sheets demonstrated only modest gains relative to the robust growth seen with the double layer variety. Together, these data verify the utility of thermoresponsive cell sheets for use in salivary glands and indicates the double layer form to provide the best option in terms of cell differentiation and recovered tissue integrity, thereby offering a potential new therapeutic strategy for treating hyposalivation.
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http://dx.doi.org/10.1038/s41536-019-0078-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609686PMC
July 2019

Utah's cell sheet tissue engineering center.

Regen Ther 2019 Dec 10;11:2-4. Epub 2019 May 10.

Cell Sheet Tissue Engineering Center (CSTEC), Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, 30 South 2000 East, Salt Lake City, UT 84112, USA.

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http://dx.doi.org/10.1016/j.reth.2019.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517791PMC
December 2019

Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis.

NPJ Regen Med 2019 21;4. Epub 2019 Feb 21.

1Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193 Japan.

Current cartilage regenerative therapies are not fully effective in treating osteoarthritis of the knee (OAK). We have developed chondrocyte sheets for autologous transplantation and tested these in in vitro and in vivo preclinical studies, and have reported that the transplantation of chondrocyte sheets promoted hyaline cartilage repair in rat, rabbit, and minipig models. However, autologous transplantation of chondrocyte sheets has yet to be reported in humans. Here, we report our combination therapy in which conventional surgical treatment for OAK, is followed by autologous chondrocyte sheet transplantation for cartilage repair. Eight patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV receive the therapy. Patients are thoroughly assessed by preoperative and postoperative X-rays, magnetic resonance imaging (MRI), arthroscopy, Knee injury and Osteoarthritis Outcome Score (KOOS), Lysholm Knee Score (LKS), and a laser-induced photoacoustic method to assess cartilage viscoelasticity. Arthroscopic biopsies of all patients are performed 12 months after transplantation for histological evaluation. The properties of the chondrocyte sheets are evaluated using gene expression analysis to investigate the ability to predict the clinical and structural outcomes of the therapy. For this small initial longitudinal series, combination therapy is effective, as assessed by MRI, arthroscopy, viscoelasticity, histology, and the clinical outcomes of KOOS and LKS. Gene marker sets identified in autologous chondrocyte sheets may be predictive of the overall KOOS, LKS, and histological scores after therapy. These predictive gene sets may be potential alternative markers for evaluating OAK treatment.
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http://dx.doi.org/10.1038/s41536-019-0069-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384900PMC
February 2019

Efficient intrahepatic tumor generation by cell sheet transplantation to fabricate orthotopic hepatocarcinoma-bearing model mice for drug testing.

J Biomed Mater Res A 2019 05 27;107(5):1071-1079. Epub 2019 Feb 27.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), Kawada-cho 8-1, Shinjuku-ku, Tokyo 162-8666, Japan.

Subcutaneous tumor-bearing mice are commonly used to evaluate antitumor activity in preclinical studies of anticancer drugs. However, these models often exhibit excessive antitumor responses to anticancer drug candidates. In this study, intrahepatic tumor-bearing mice as orthotopic tumor models were fabricated by transplanting hepatocarcinoma cell monolayers (sheets) to investigate differences in ectopic versus orthotopic antitumor response. Cell sheets, harvested from temperature-responsive cell culture dishes using thin gelatin gel supporters, were transferred onto mouse liver surfaces. Cell sheet transplantation drastically improved intrahepatic tumor formation compared with direct intrahepatic injection of dispersed cells. In particular, all cell sheet-transplanted mice formed well-developed tumors inside the liver following removal of the mesothelial membrane at the liver surface. Notably, these mice exhibited comparable life spans, indicating similar intrahepatic tumor development rates. Antitumor activity of doxorubicin (DOX) was examined using both subcutaneous and intrahepatic tumor-bearing mice. Although DOX administration yielded decreased subcutaneous tumor volumes, intrahepatic tumors exhibited no significant antitumor response. The results were considered to represent pharmacokinetic and histological structure differences between ectopic and orthotopic tumors, and partially supported the clinical uses of DOX. Therefore, cancer cell sheet transplantation constitutes a promising method to fabricate intrahepatic tumor-bearing mice for drug screening test in preclinical studies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1071-1079, 2019.
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http://dx.doi.org/10.1002/jbm.a.36641DOI Listing
May 2019

Cell sheet tissue engineering: Cell sheet preparation, harvesting/manipulation, and transplantation.

J Biomed Mater Res A 2019 05 21;107(5):955-967. Epub 2019 Feb 21.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan.

Cell sheet tissue engineering is a concept for creating transplantable two-dimensional (2D) and three-dimensional (3D) tissues and organs. This review describes three elements of cell sheet tissue engineering in terms of the chemical and physical effects of material surfaces and the interfacial properties of cell sheets: preparation, harvesting/manipulation and transplantation of cell sheets. An essential technology for the preparation of cell sheets is the use of a temperature-responsive cell culture surface, where the surface of tissue culture polystyrene (TCPS) dish is modified with thin layer of temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). PIPAAm-immobilized TCPS allows cultured cells to be harvested as a contiguous cell sheet with extracellular matrices (ECMs) by reducing the temperature, while chemical and physical disruption impair ECMs, cell-cell junction, and membrane proteins. Ligand-immobilized and porous hydrophilic PIPAAm-grafted surfaces are able to accelerate cell sheet preparation and harvesting, respectively. In addition, the manipulation of harvested cell sheets with the aid of cell sheet manipulator facilitates the formation of 3D tissues. Cell sheet-based tissues and their transplantation are in seven clinical settings such as heart, cornea, esophagus, periodontal, middle chamber of ear, knee cartilage, and lung. In order to create thick and large 3D tissues and organs, large production of differentiated parenchymal cells from induced pluripotent stem (iPS) cells and vascularization within 3D tissues are key issues. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 955-967, 2019.
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http://dx.doi.org/10.1002/jbm.a.36627DOI Listing
May 2019

Allogeneic multipotent mesenchymal stromal cell sheet transplantation promotes healthy healing of wounds caused by zoledronate and dexamethasone in canine mandibular bones.

Regen Ther 2019 Jun 12;10:77-83. Epub 2019 Jan 12.

Department of Oral and Maxillofacial Surgery, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

Introduction: Many cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ), which is an intractable disease, have been reported. Although a general intravenous injection of multipotent mesenchymal stromal cells (MSCs) may be effective for treating BRONJ, it has some severe problems. Therefore, our aim was to develop a treatment of locally administered MSCs. In this study, we investigated the effect of MSC sheet transplantation in the mandibular bone healing in beagle dogs, which were administered zoledronate and dexamethasone.

Methods: MSCs isolated from subcutaneous fat were seeded onto temperature-responsive culture dishes to produce MSC sheets. Zoledronate and dexamethasone were administered to beagle dogs. Then, the parts of mandibular cortical bones were removed, and MSC sheets were transplanted to cover those bone defects (MSC sheet transplant side) or not (Control side). The specimens were evaluated in micro CT, histology, and immunohistochemistry.

Results: Four weeks after surgery, redness and swellings were observed in the mucosal wounds of the control sides of 2 of 3 dogs. In contrast, the mucosal wounds of the MSC sheet transplant sides of all dogs completely healed. Histological images showed some free sequestrums and many bacterial colonies, and Immunohistological analysis showed some cathepsin K-positive multinuclear cells detached from jaw bone surfaces in the control sides.

Conclusions: MSC sheet transplantation promotes healthy healing of wounds caused by zoledronate and dexamethasone in canine mandibular bones. And the injured canine mandibular bones administered zoledronate and dexamethasone showed BRONJ-like findings.
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http://dx.doi.org/10.1016/j.reth.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330512PMC
June 2019