Thermoreversible Gelation Polymer Research Articles

MP22-11 SUCCESSFUL TRANSURETHRAL ENGRAFTMENT OF BUCCAL MUCOSAL CELLS EXPANDED IN VITRO, TRANSPLANTED IN A POLYMER SCAFFOLD CARRIER IN A RABBIT URETHRAL STRICTURE MODEL, AND PROVEN WITH IMMUNOHISTOCHEMICAL BIOMARKERS

You have accessJournal of UrologyCME1 May 2022MP22-11 SUCCESSFUL TRANSURETHRAL ENGRAFTMENT OF BUCCAL MUCOSAL CELLS EXPANDED IN VITRO, TRANSPLANTED IN A POLYMER SCAFFOLD CARRIER IN A RABBIT URETHRAL STRICTURE MODEL, AND PROVEN WITH IMMUNOHISTOCHEMICAL BIOMARKERS Akio Horiguchi, Toshihiro Kushibiki, Kenichiro Ojima, Masayuki Shinchi, Yusuke Hirano, Yoshine Mayumi, Keiichi Ito, Kosuke Miyai, Ichiro Miura, Masayuki Takeda, Masaru Iwasaki, Shojiro Kato, Senthilkumar Rajappa, and Samuel J.K. Abraham Akio HoriguchiAkio Horiguchi More articles by this author , Toshihiro KushibikiToshihiro Kushibiki More articles by this author , Kenichiro OjimaKenichiro Ojima More articles by this author , Masayuki ShinchiMasayuki Shinchi More articles by this author , Yusuke HiranoYusuke Hirano More articles by this author , Yoshine MayumiYoshine Mayumi More articles by this author , Keiichi ItoKeiichi Ito More articles by this author , Kosuke MiyaiKosuke Miyai More articles by this author , Ichiro MiuraIchiro Miura More articles by this author , Masayuki TakedaMasayuki Takeda More articles by this author , Masaru IwasakiMasaru Iwasaki More articles by this author , Shojiro KatoShojiro Kato More articles by this author , Senthilkumar RajappaSenthilkumar Rajappa More articles by this author , and Samuel J.K. AbrahamSamuel J.K. Abraham More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002561.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Buccal mucosal (BM) cell therapies with different processing methods have been developed to treat male urethral stricture (US). Our earlier studies with six human subjects (DOI: 10.1111/iju.13852) and an animal model (DOI: 10.1016/j.reth.2021.05.004) confirmed the transurethral engraftment of autologous BM cells at urethral stricture sites when transplanted after encapsulation in a thermoreversible gelation polymer (TGP) scaffold. We report the reproducibility of the procedure in rabbit US models, proving engraftment with an immunohistochemical analysis. METHODS: The urethra of six male Japanese white rabbits were electro coagulated; US development was confirmed by endoscopy and urethrography 4 weeks later. The BM tissue was harvested, and its epithelial cells isolated and cultured for 7 days. After incising the US with a urethrotome, an 8 Fr-Nelaton catheter was placed in the urethra, and cultured epithelial cells suspended in TGP were injected into it. On the third day, the catheter was removed. On the seventh day after transplantation, the urethra was harvested and subjected to conventional hematoxylin and eosin (HE) staining and immunostaining with an anti-cytokeratin 14 (CK14) antibody (a marker for BM epithelial cells) and anti-GATA binding protein 3 (GATA3) antibody (a marker for urothelium). RESULTS: All animals developed US at the electrocoagulation site. HE staining showed that the urethral mucosal defect caused by the urethrotomy was covered with a multilayered squamous epithelium in all rabbits (Fig. 1A and D). The CK14 was positive on the lumen covered with a squamous epithelium (Fig. 1B and E). The GATA3 was negative in the region positive for CK14 while positively staining the remaining urothelium (Fig. 1C and F)). CONCLUSIONS: Autologous BM cells with highly suggestive cell engraftment at the urethrotomy site based on immunohistochemical biomarkers are worth considering for a larger clinical study in male US. Source of Funding: none © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e375 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akio Horiguchi More articles by this author Toshihiro Kushibiki More articles by this author Kenichiro Ojima More articles by this author Masayuki Shinchi More articles by this author Yusuke Hirano More articles by this author Yoshine Mayumi More articles by this author Keiichi Ito More articles by this author Kosuke Miyai More articles by this author Ichiro Miura More articles by this author Masayuki Takeda More articles by this author Masaru Iwasaki More articles by this author Shojiro Kato More articles by this author Senthilkumar Rajappa More articles by this author Samuel J.K. Abraham More articles by this author Expand All Advertisement PDF DownloadLoading ...

An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing

BackgroundChondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco's Modified Eagle's Medium (DMEM) are commonly employed at 4–8 °C. MethodsIn this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (∼25 °C) and ii) ECS transport at 4 °C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. ResultsThe cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. ConclusionThis study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

In Vitro Culture Expansion and Characterization of Buccal Mucosal Epithelial Cells for Tissue Engineering Applications in Urethral Stricture After Transportation Using a Thermoreversible Gelation Polymer.

Introduction: The transportation of tissues from hospitals to clinical laboratories for cell therapy is an essential component of regenerative medicine. Previously, we used laboratory-cultured mucosal cells from buccal epithelium expanded and encapsulated using a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) procedure. In this study, to improve the outcomes, we compared the thermoreversible gelation polymer (TGP) transportation procedure with conventional culture methods, and reported its advantages. Methods: Human buccal mucosal tissues in Phase I of the study were transported in Euro-Collins solution (ECS) and the cells obtained were cultured in two-dimensional (2D) Dulbecco's modified Eagle's medium (DMEM), CnT-Prime epithelial 2D differentiation medium (CnT-PR), and a three-dimensional (3D)-TGP scaffold. In Phase II, tissues were transported in a TGP cocktail and the ECS. The cells were cultured in 2D-DMEM and 3D-TGP, quantified, and characterized by immunohistochemistry. Results: The cells in 3D-TGP culture maintained epithelial morphology in a better manner compared with 2D-DMEM, in which they developed fibroblast-like morphology. The TGP-transported cells grew rapidly. Immunohistochemical analysis results for AE1/AE3, EGFR, integrin-β1, p63, and p75 were intensely positive in 3D-TGP. Conclusion: The TGP-based cocktail used in human buccal tissue transportation yielded cells with better morphology maintenance. The TGP scaffold provides an optimal in vitro environment wherein epithelial cells better maintain their native phenotype compared to those cultured through conventional methods. These results suggest using TGP for the transportation and culture of human buccal tissues for clinical applications. In addition, the use of a TGP-based cocktail for the transport of other tissues for regenerative medicine applications is worth further analysis.

Organoid culture of mouse fallopian tube epithelial stem cells with a thermo-reversible gelation polymer

In this study, a three-dimensional (3D) thermo-reversible gelation polymer (TGP) culture system was established for organoid culture of mouse fallopian tube (FT) epithelial stem cells (FTESCs) without cell isolation. FT tissues from 6- to 8-week-old ICR mice were digested with collagenase, and whole FT cells (FTCs) were inoculated into the TGP. After 6 days of culture, many spheres in the TGP formed. Some cells in the spheres were positive for 5-ethynyl-2′-deoxyuridine (EdU), a marker of cell proliferation. Furthermore, all the spheres that formed in the TGP were also labelled for EpCAM and LGR5. Some cells in the spheres were stained for PAX8, a secretory cell marker, and fewer cells were labelled with TUBB4, a ciliated cell marker. These results indicate that the 3D TGP culture system is a useful tool for organoid culture of FTESCs in vitro.

Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

Regenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

Enhanced miRNA-140 expression of osteoarthritis-affected human chondrocytes cultured in a polymer based three-dimensional (3D) matrix

AimsWe have evaluated the potential of a three-dimensional (3D) thermoreversible gelation polymer (TGP) matrix in enhancing miRNA 140 expression (a biomarker correlating with homeostasis and cartilage regeneration) during the in vitro expansion of osteoarthritis (OA)-affected human chondrocytes. Materials and methodsOA-chondrocytes were cultured in two-dimensional (2D) monolayer followed by culture in 3D-TGP. miRNA 140 expression levels in cell culture supernatant followed by expression in the cell lysate of both 2D and 3D-TGP cultures were analyzed. Key findingsThe expression of miRNA 140 in cell culture supernatant from the 3D-TGP group was 0.001 to 0.002% that in 2D culture supernatant while in the cell lysate, miRNA 140 expression in the 3D-TGP was nearly 30-fold higher than that of 2D group. SignificanceThe 3D-TGP matrix allows enhanced expression of miRNA 140 in OA-affected human chondrocytes in vitro which after necessary validations can be applied in clinical transplantation to significantly improve the outcome.

A novel human donor cornea preservation cocktail incorporating a thermo-reversible gelation polymer (TGP), enhancing the corneal endothelial cell density maintenance and explant culture of corneal limbal cells

PurposeMcCarey-Kaufman’s (MK) medium and Optisol-GS medium are the most commonly employed media for human donor corneal preservation. In this study, we evaluated the preservation efficacy of discarded human donor corneas using a Thermo-reversible gelation polymer (TGP) added to these two media.MethodsThirteen human corneal buttons collected from deceased donors, which were otherwise discarded due to low endothelial cell density (ECD) were used. They were stored in four groups: MK medium, MK medium with TGP, Optisol-GS and Optisol-GS with TGP at 4 °C for 96 h. Slit lamp examination and specular microscopy were performed. Corneal limbal tissues from these corneas were then cultured using explant methodology one with and the other without TGP scaffold, for 21 days.ResultsMK + TGP and Optisol-GS + TGP preserved corneas better than without TGP, which was observed by maintenance of ECD which was significantly higher in Optisol-GS + TGP than MK + TGP (p-value = 0.000478) and corneal thickness remaining the same for 96 h. Viable corneal epithelial cells could be grown from the corneas stored only in MK + TGP and Optisol-GS + TGP. During culture, the TGP scaffold helped maintain the native epithelial phenotype and progenitor/stem cell growth was confirmed by RT-PCR characterization.ConclusionTGP reconstituted with MK and Optisol—GS media yields better preservation of human corneal buttons in terms of relatively higher ECD maintenance and better in vitro culture outcome of corneal limbal tissue. This method has the potential to become a standard donor corneal transportation-preservation methodology and it can also be extended to other tissue or organ transportation upon further validation.

Enhanced expression of hyaluronic acid in osteoarthritis-affected knee-cartilage chondrocytes during three-dimensional in vitro culture in a hyaluronic-acid-retaining polymer scaffold

BackgroundChondrocyte transplantation to address cartilage damage is an established solution. Because hyaluronic acid (HA) is an essential component for homeostasis of the cartilage, in order to arrive at methodologies to utilize its advantages in cell-based therapies, we compared the HA retention capability of a thermoreversible gelation polymer scaffold-based environment (3D-TGP) with conventional in vitro cell culture methodologies. MethodsChondrocytes derived from osteoarthritis-affected knee joint cartilage of elderly patients were used and accomplished in three phases. In Phase I, the levels of HA secreted by chondrocytes were measured in culture supernatant. In Phase II, retention capacity of externally added HA was quantified indirectly by measuring the HA released in culture supernatant, and in Phase III, the expression of CD44 on cells was analysed by immunohistochemistry. ResultsIn Phase I, the average HA in the 3D supernatant was 3% that of 2D. In phase II, 80% of externally added HA was detected in the 2D on day 7, while in 3D-TGP, only 0.1% was released until day 21. In Phase III, 2D yielded individual cells that started degenerating from the third week; in 3D-TGP cells grew for a longer duration, formed a tissue-like architecture with extracellular matrix with significantly intense staining of CD44 than 2D. ConclusionThe capability of the 3D-TGP culture environment to retain HA and support chondrocytes to grow with a tissue-like architecture expressing higher HA content is considered advantageous as it serves as an in vitro culture platform that enables tissue engineering of cartilage tissue with native hyaline phenotype and higher HA expression. The in vitro environment being conducive, based on this data, we also recommend that the TGP be tried as an encapsulation material in clinical studies of chondrocyte implantation for optimal clinical outcome.

CREATING CORNEAL STEM CELLS WITH A THEROREVERSIBLE GELATION POLYMER

Introduction: Recent technological advancements in single-cell RNA sequencing and single-cell quantitative real-time PCR have enabled researchers to conduct single-cell analyses of limbal epithelial cells. Aim of the study: the main aim of the study is to Cultivating corneal stem cells with a thermoreversible gelation polymer Material and method: Each human limbal biopsy were placed in 1 mL of TC medium containing 3% FCS, antibiotics (penicillin, gentamicin, and amphotericin B), and DMEM, and then transported to a cell biology lab. Conclusion: The thermoreversible gelation polymer facilitates the growth of limbal epithelial cells. The limbal characteristic could still be seen in the grown cells

A three-dimensional in vitro culture environment of a novel polymer scaffold, yielding chondroprogenitors and mesenchymal stem cells in human chondrocytes derived from osteoarthritis-affected cartilage tissue

ObjectiveWe evaluated the expression of stem/progenitor biomarkers in osteoarthritic tissue derived chondrocytes cultured using a three-dimensional (3D) thermo-reversible gelation polymer (TGP). MethodsThe chondrocytes from discarded biopsy tissues obtained from human elderly patients with osteoarthritis were cultured using the 3D-TGP up to six weeks. ResultsThe chondrocytes grew in a tissue-like manner, without de-differentiation into fibroblasts, and the cells thus tissue-engineered were proven positive for CD49e, OCT4, CD-105 and STRO-1 by immunohistochemistry. ConclusionThis study establishes the efficacy of this 3D-TGP platform for clinically useable in-vitro tissue-engineered cartilage for improvising the clinical outcome of cell therapy for cartilage repair.

Articular chondrocytes from osteoarthritic knee joints of elderly, invitro expanded in thermo-reversible gelation polymer (TGP), exhibiting higher UEA-1 expression in lectin microarray.

Autologous chondrocytes in vitro expanded, are used as tools of regenerative therapies for cartilage injuries. However, inability to maintain the hyaline phenotype both in vitro and post in vivo transplantation, remains one of the major hurdles for long term efficacy under clinical settings. We have reported earlier, hyaline phenotype maintenance of both human and rabbit chondrocytes for a long duration both in vitro when cultured conditions using a Thermo-reversible Gelation Polymer (TGP) scaffold-based methodology and in vivo post-transplantation animal model of cartilage damage. Having intrigued by such encouraging outcome, we in this study, analysed the similar TGP culture environment whether would be able to allow in vitro expansion of severe osteoarthritis affected cartilage tissue from elderly patients and evaluated the cells using lectin microarray characterization for pluripotency. Cartilage tissue were obtained from patients (n = 7; age: 60–85 years) undergoing total knee arthroplasty for severe osteoarthritis. Chondrocytes were isolated and cultured in two groups: i. conventional culture without scaffold (2D) and ii. using a TGP scaffold-based culture (3D) up to 18 weeks. In addition to earlier reported findings such as maintenance of hyaline phenotype having been confirmed in this study as well, surface glycoprotein analysis by lectin microarray demonstrated that the α1-2 Fuc recognition lectin (UEA-1) (marker reported in literature for pluripotent stem cells) was found to be more highly expressed in 3D culture compared to 2D culture and even increased over time in 3D culture. We have developed an environment where osteoarthritis affected chondrocytes from the elderly could be cultured up to 18 weeks in vitro using TGP scaffold which express pluripotent cell associated surface glycoproteins compared to the conventional methodology.

An Improved Encapsulation Method for Cryopreserving Hepatocytes for Functional Transplantation Using a Thermo-reversible Gelation Polymer.

Thermo-reversible gelation polymer (TGP) can be converted into a gel state upon warming and liquid upon cooling. The present study aimed to demonstrate a new method for cryopreservation and encapsulation of rat hepatocytes using a TGP and their successful transplantation. The isolated rat hepatocytes were microencapsulated using TGP, and stored in liquid nitrogen. After cryopreservation, hepatocytes were cultured. Moreover, hepatocytes were transplanted into the spleen without a TGP capsule. The viability of hepatocytes that were cryopreserved in TGP was 71.2±2.3%. The hepatocytes demonstrated adequate survival, maintained their hepatic function in culture, and expressed albumin after transplantation to the rat spleen. We demonstrated a cryopreservation method of rat hepatocyte encapsulation using a TGP gel in the hydrogel state which subsequently allowed successful transplantation of unencapsulated hepatocytes in a sol state TGP gel at low temperature.

Buccal epithelium Expanded and Encapsulated in Scaffold-Hybrid Approach to Urethral Stricture (BEES-HAUS) procedure: A novel cell therapy-based pilot study.

ObjectivesTo describe the feasibility of a novel cell‐based endoscopic technique using buccal epithelium, expanded and encapsulated in a thermoreversible gelation polymer scaffold for the treatment of urethral stricture.MethodsSix male patients with bulbar urethral stricture ranging from 2.0 to 3.5 cm in length were included in this pilot study. Autologous buccal epithelial cells from a small buccal mucosal biopsy were isolated, cultured and encapsulated in thermoreversible gelation polymer scaffold, and were implanted at the stricture site after a wide endoscopic urethrotomy.ResultsAll the patients voided well, with a mean peak flow rate of 24 mL/s. Urethroscopy carried out at 6 months showed healthy mucosa at the urethrotomy site. However, two of the six patients had recurrence at 18 and 24 months, respectively.ConclusionsThis endoscopic‐based Buccal epithelium Expanded and Encapsulated in Scaffold‐Hybrid Approach to Urethral Stricture (BEES‐HAUS) technique is a promising alternative for the open substitution buccal graft urethroplasty. It is possible to achieve the benefits of open substitution buccal urethroplasty with this endoscopic technique.

Characterization and application of iPSCs in drug discovery research. Focus in hepatocytes

Purpose: A novel three dimensional (3D) culture system purely synthesised from co-polymer which is free from biological contamination for Huh7 cell cultivation and hepatitis C virus (HCV) replication has been attempted. Materials and Methods: Mebiolgel, a thermo-reversible gelation polymer was used as a 3D scaffold for culturing Huh7, a liver carcinoma cell line used in our study. The 3D culture of the cells were infected with cell culture derived HCV. Result: The scaffold supported the cell growth as 3D spheroids for up to 63 days. Moreover mebiolgel was found to be improving the hepatocyte differentiation of Huh7 cells at the transcript level. Three dimensional culture was susceptible for HCV infection, and this was confirmed by detecting the HCV replication intermediate viral core antigen. Conclusion: Mebiolgel based culture system was proven to be suited for 3D culture of Huh7 cells by improvising liver specific genotypic expression and was susceptible for HCV replication. Since mebiolgel based Huh 7 express better hepatocyte differentiation markers genotypically, this can be implemented as an alternate for primary hepatocytes in studies such as viral isolation from patient serum.

Transplantation of autologous chondrocytes ex-vivo expanded using Thermoreversible Gelation Polymer in a rabbit model of articular cartilage defect

Graft failure due to de-differentiation of the chondrocytes during in vitro culture and after transplantation is a major hurdle in Autologous Chondrocyte Implantation (ACI). We, herein, report the transplantation of autologous chondrocytes ex vivo expanded using a Thermo-reversible Gelation Polymer (TGP) in a rabbit model. A full thickness chondral defect was created in one of the knee joints in each of the six rabbits of the study and autologous chondrocytes in vitro expanded using TGP scaffold were transplanted after 10 weeks. H & E staining of the biopsy after 6 months revealed maintenance of articular cartilage phenotype.

Usefulness of the Behavior of Fibroblast Attachment to Coils in Thermoreversible Gelation Polymer for Aneurysmal Coil Treatment

Background: Post embolization aneurysm reccurence is still a drawback in endovascular coiling for cerebral aneuryms. That stimulated reaearch into new promising embolic materials, as the Thermoreversible Gelation Polymer (TGP) when used as cell culture medium with its temperature related properties. We evaluated proliferative properties of fibroblast cell cultures in association with metalic coils. Methods: Intial assessment of cell viability was done for 3T3 (mouse fibroblast) and A172 (glioma cell) lines for 6 and 16 days in TGP solid phase and media. After that TGP was liquified and cell attachment confirmed. Next, fibroblasts were cultivated under the same conditions adding Guglielmi detachable coils for 24 h and 3 days with TGP, evaluating cell attachment and proliferation on coils. Last, the time course of cell proliferation was evaluated, comparing cell numbers on equal coil surface areas for 1,3,5,7,10,14 and 21 days in TGP. Electron microscopy was used for cell assessment on the coils. Results: Fibroblasts survive and show satisfactory viability in TGP. In liquefied TGP after 6 days and 16days, both cell types temporally extended processes and attached on flask`s bottom. Next after 24 h and 3 days fibroblast cultivation with coils in TGP, cells extended processes and attached to coils, indicating some proliferation and clustering. Last, the day 1 attached cell numbers on coils cultivated in TGP gradually decreased until day 10 and later increased again to just below day 1 level. However, both fibroblast and glioma cells in TGP did not extend processes, move or proliferate, and had spherical shape while in solid TGP. Conclusion: Fibroblasts attachment and long survival on coils in TGP may facilitate aneurysm treatment to achieve better volume embolization rate and improve intra-aneurysmal thrombus organization.

Mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of Huh7 cell line with improved hepatocyte differentiation marker expression and HCV replication

Purpose: A novel three dimensional (3D) culture system purely synthesised from co-polymer which is free from biological contamination for Huh7 cell cultivation and hepatitis C virus (HCV) replication has been attempted. Materials and Methods: Mebiolgel, a thermo-reversible gelation polymer was used as a 3D scaffold for culturing Huh7, a liver carcinoma cell line used in our study. The 3D culture of the cells were infected with cell culture derived HCV. Result: The scaffold supported the cell growth as 3D spheroids for up to 63 days. Moreover mebiolgel was found to be improving the hepatocyte differentiation of Huh7 cells at the transcript level. Three dimensional culture was susceptible for HCV infection, and this was confirmed by detecting the HCV replication intermediate viral core antigen. Conclusion: Mebiolgel based culture system was proven to be suited for 3D culture of Huh7 cells by improvising liver specific genotypic expression and was susceptible for HCV replication. Since mebiolgel based Huh 7 express better hepatocyte differentiation markers genotypically, this can be implemented as an alternate for primary hepatocytes in studies such as viral isolation from patient serum.

A new system to evaluate the influence of immunosuppressive drugs on pancreatic islets using epigenetic analysis in a 3-dimensional culture.

The present study aimed to establish a new method to evaluate the influence of immunosuppressive drugs on pancreatic islets in short-term in vitro cultures using epigenetic analysis in a 3-dimensional culture. For this purpose, we selected (a) a 3-dimensional culture system utilizing thermoreversible gelation polymer, (b) pancreatic duodenal homeobox-1 (pdx-1)-Venus transgenic pigs expressing the green fluorescent protein, (c) FK506 as an immunosuppressive drug of the evaluation, and (d) the bisulfite sequencing technique to evaluate the methylation levels of pdx-1 and insulin genes. Each isolated pancreatic islet was cultured with several doses of FK506. The viability of the each islet was evaluated by analyzing the emission of Venus in real time and by propidium iodide staining. Epigenetic analysis was performed at several time points. Each single pancreatic islet was stably cultured for 30 days in this system. At day 30 in culture, we observed that insulin DNA methylation levels in the group that received a high dose of FK506 dramatically increased, although there was no change in pdx-1 DNA methylation level and configuration of the islets. Our system may be useful to determine immunosuppressive drugs that are specifically suitable for islet transplantation.

Successful transportation of human corneal endothelial tissues without cool preservation in varying Indian tropical climatic conditions and in vitro cell expansion using a novel polymer

Background:Though the transplantation of human corneal endothelial tissue (CET) separated from cadaver cornea is in practice, its transportation has not been reported. We report the successful transportation of CET in varying Indian climatic conditions without cool preservation and the in vitro expansion of Human Corneal Endothelial Precursor Cells (HCEPCs) using a novel Thermo-reversible gelation polymer (TGP).Materials and Methods:CET from cadaver corneas (n = 67), unsuitable for transplantation, were used. In phase I, CET was transported in Basal Culture Medium (Group I) and TGP (Group II) and in Phase II, in TGP cocktail alone, from three hospitals 250-2500 km away, to a central laboratory. The transportation time ranged from 6 h to 72 h and the outdoor temperature between 20°C and 41°C. On arrival, CET were processed, cells were expanded upto 30 days in basal culture medium (Group A) and TGP scaffold (Group B). Cell viability and morphology were documented and Reverse transcription polymerase chain reaction (RT-PCR) characterization undertaken.Results:In Phase I, TGP yielded more viable cells (0.11 × 106 cells) than Group I (0.04 × 106 cells). In Phase II, the average cell count was 5.44 × 104 cells. During expansion, viability of HCEPCs spheres in TGP was maintained for a longer duration. The cells from both the groups tested positive for B-3 tubulin and negative for cytokeratins K3 and K12, thereby proving them to be HCEPCs.Conclusion:TGP preserves the CET during transportation without cool preservation and supports in vitro expansion, with a higher yield of HCEPCs, similar to that reported in clinical studies.

3P318 温度感受性ゲルを用いた蛍光検出によるドロップレットソーティング技術の開発とその応用(28. バイオエンジニアリング,ポスター,第52回日本生物物理学会年会(2014年度))

3P318 温度感受性ゲルを用いた蛍光検出によるドロップレットソーティング技術の開発とその応用(28. バイオエンジニアリング,ポスター,第52回日本生物物理学会年会(2014年度))