Beneficial immune-modulatory effects of the N-163 strain of Aureobasidium pullulans-produced 1,3-1,6 Beta glucans in Duchenne muscular dystrophy: Results of an open-label, prospective, exploratory case-control clinical study.

Background: This exploratory case-control study is to evaluate the effects of supplementation of Aureobasidium pullulans-N-163 strain produced 1,3-1,- 6 beta glucan in young patients with Duchenne muscular dystrophy (DMD). Methods: Twenty-seven male subjects aged 5-19 years with DMD were included, nine in the control arm and 18 in the treatment arm to receive N-163 beta glucan along with conventional therapies for 45 days. While performing the analysis, steroid usage was also taken into consideration, those not administered steroids (Steroid -ve) (Control, n = 5; treatment, n = 9), those administered steroids (Steroid +ve) (Control, n = 4; treatment, n = 9). Results: IL-6 showed a significant decrease in the treatment groups, especially the N-163 Steroid -ve group. IL-13 decreased in both treatment groups and TGF-ß levels showed a significant decrease in the treatment groups, especially the N-163 Steroid -ve group, (p < 0.05). Dystrophin levels increased by up to 32% in the treatment groups compared to the control. Medical research council (MRC) grading showed slight improvement in muscle strength improvement in 12 out of 18 patients (67%) in the treatment group and four out of nine (44%) subjects in the control group. Conclusion: Supplementation with the N-163 beta glucan food supplement produced beneficial effects: a significant decrease in inflammation and fibrosis markers, increase in serum dystrophin and slight improvement in muscle strength in DMD subjects over 45 days, thus making this a potential adjunct treatment for DMD after validation. Trial registration: The study was registered in Clinical trials registry of India, CTRI/2021/05/033346. Registered on 5th May, 2021.

Resolution of fibrosis in mdx dystrophic mouse after oral consumption of N-163 strain of Aureobasidium pullulans produced ß-glucan.

Recent advances in the management of Duchenne muscular dystrophy (DMD), such as exon skipping and gene therapy, though have reached a clinical stage, the outcome at its best is still considered suboptimal. In this study, we evaluated a novel N-163 strain of Aureobasidium pullulans produced ß-glucan (Neu-REFIX) for its potential as an adjuvant to slow down the progression of the disease by anti-inflammatory and anti-fibrotic effects. In this study, 45 mice in the three groups, 15 each in a group; Gr. 1 normal mice, Gr.2 mdx mice as vehicle, and Gr.3 mdx mice administered the N-163 ß-glucan for 45 days. The N-163 ß-glucan group showed a significant decrease in the plasma ALT, AST, and LDH levels (126 ± 69 U/l, 634 ± 371 U/l, 3335 ± 1258 U/l) compared with the vehicle group (177 ± 27 U/l, 912 ± 126 U/l, 4186 ± 398 U/l). Plasma TGF-ß levels increased, and plasma IL-13 levels decreased in the N-163 group. The inflammation score of HE-stained muscle sections in the N-163 group (1.5 ± 0.8) was lower than that in the vehicle group (2.0 ± 0.8). The N-163 strain ß-glucan group (24.22 ± 4.80) showed a significant decrease in the fibrosis area (Masson's Trichrome-positive area) compared with the vehicle group (36.78 ± 5.74). The percentage of centrally nucleated fibres evaluated by Masson's trichrome staining was 0 in the normal group, while it increased to 80% in the vehicle group but remained at 76.8% in the N-163 group. The N-163 ß-glucan group showed a significant decrease in the fibrosis area. Considering their safety and easy oral consumption, Neu-REFIX ß-glucan could be worth large multicentre clinical studies as adjuvant in slowing down the progress of DMD.

Beneficial effects of 1,3-1,6 ß-glucans produced by Aureobasidium pullulans on non-esterified fatty acid levels in diabetic KKAy mice and their potential implications in metabolic dysregulation.

Objectives: In this study, we used an obese and diabetic mouse model to compare two strains of Aureobasidium pullulans (AFO-202 and N-163) produced beta-glucans (ß-glucans), which alleviate lipotoxicity. Methods: Four groups of KK-Ay mice were used, with six subjects in each group. Group 1: sacrificed on day 0 for baseline values; Group 2: control (drinking water); Group 3: AFO-202 beta glucan-200 mg/kg/day; Group 4: N-163 beta glucan-300 mg/kg/day for 28 consecutive days. Results: Group 4 (N-163) had the lowest non-esterified fatty acids (NEFA) levels and marginally decreased triglyceride levels compared to the other groups. There were no significant differences in blood glucose, hemoglobin A1c (HbA1c), triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol levels. N-163 ß-glucans decreased NEFA levels after 28 days. Conclusion: These results, although modest, warrant further in-depth research into lipotoxicity and associated inflammatory cascades in both healthy and diseased subjects for the prevention and management of metabolic dysregulation and associated diseases such as non-alcoholic fatty liver disease (NAFLD).

Engraftment and Proliferation of Thermoreversible-Gelation-Polymer-Encapsulated Human Corneal Limbal-Stem-Cells on Ocular Surface of a Cadaver Cornea.

PURPOSE: Corneal limbal stem cell (LSC) transplantation has been reported as a potential approach to treat the damaged corneal epithelium. Scaffolds such as human amniotic membrane (hAM) are commonly employed for the in vitro culture and as a carrier during in vivo transplantation. However, they carry the risk of biological contamination and donor to donor variability. To overcome these disadvantages, we herein report the capabilities of a synthetic thermoreversible gelation polymer (TGP) scaffold to serve as an encapsulation support during LSC transplantation and to enable engraftment for corneal regeneration. METHODS: Sixteen discarded human corneas were used to isolate the corneal epithelium which was cultured in TGP and hAM. The cell proliferation and characteristics between TGP and hAM culture methods were evaluated by microscopic observation, 3H Thymidine incorporation assay, immunoperoxidase and immunofluorescence staining. RESULTS: The 3H Thymidine assay's results showed that TGP allowed human-donor cornea-derived LSCs to proliferate well in vitro, compared to hAM and the cells encapsulated in TGP and transplanted ex vivo onto a human cadaver donor cornea denuded of its epithelium, migrated on the ocular surface, and proliferated to form a continuous layer in 25 days. Immunoperoxidase and Immunofluorescence staining of TGP-cultured cells were positive for LSC markers (p63, ABCG2, Connexin 43 and Integrin ß), proving that the TGP helps to preserve the limbal cells' stemness. CONCLUSION: TGP is found to be a multipurpose scaffold for (i) in vitro culture, (ii) ex vivo encapsulation, and in vivo transplantation (iii), enabling engraftment of LSCs in this study, with potentials to extend its application in cell-based therapies in several regenerative medicine approaches.

Benefits of Gut Microbiota Reconstitution by Beta 1,3-1,6 Glucans in Subjects with Autism Spectrum Disorder and Other Neurodegenerative Diseases.

BACKGROUND: Aureobasidium pullulans (black yeast) AFO-202 strain-produced beta glucan, Nichi Glucan, has been shown to improve the behavior and sleep pattern along with an increase in α-synuclein and melatonin in children with autism spectrum disorder (ASD). OBJECTIVE: In this randomized pilot clinical study, we have evaluated the gut microbiota of subjects with ASD after consumption of Nichi Glucan. METHODS: Eighteen subjects with ASD were randomly allocated: six subjects in the control group (Group 1): conventional treatment comprising remedial behavioral therapies and L-carnosine 500 mg per day, and 12 subjects (Group 2) underwent supplementation with Nichi Glucan 0.5 g twice daily along with the conventional treatment for 90 days. RESULTS: Whole genome metagenome (WGM) sequencing of the stool samples at baseline and after intervention showed that among genera of relevance, the abundance of Enterobacteriaceae was decreased almost to zero in Group 2 after intervention, whereas it increased from 0.36% to 0.85% in Group 1. The abundance of Bacteroides increased in Group 1, whereas it decreased in Group 2. The abundance of Prevotella increased while the abundance of Lactobacillus decreased in both Group 1 and Group 2. Among species, a decrease was seen in Escherichia coli, Akkermansia muciniphila CAG:154, Blautia spp., Coprobacillus sp., and Clostridium bolteae CAG:59, with an increase of Faecalibacterium prausnitzii and Prevotella copri, which are both beneficial. CONCLUSION: AFO-202 beta 1,3-1,6 glucan, in addition to balancing the gut microbiome in children with ASD and its role in effective control of curli-producing Enterobacteriaceae that leads to α-synuclein misfolding and accumulation, may have a prophylactic role in Parkinson's and Alzheimer's diseases as well.

Efficacy of N-163 beta-glucan in beneficially improving biomarkers of relevance to muscle function in patients with muscular dystrophies in a pilot clinical study.

Background: Muscular dystrophies other than Duchenne muscular dystrophy (DMD) are genetic diseases characterized by increasing muscle weakness, loss of ambulation, and ultimately cardiac and respiratory failure. There are currently no effective therapeutics available. Having demonstrated the efficacy of a N-163 strain of Aureobasidium Pullulans (Neu-REFIX) produced B-1, 3-1,6-Glucan in pre-clinical and clinical studies of Duchenne muscular dystrophy (DMD) earlier, we assessed the effectiveness of this novel Beta glucan in the other muscular dystrophies in the present study. Methods: In this 60-day study, six patients with muscular dystrophies other than DMD consumed one 8g gel of Neu-REFIX beta-glucan along with their usual standard of care treatment regimen, and their biomarkers of relevance to muscle function such as serum calcium (SC), creatine phosphokinase (CPK), and alkaline phosphatase (ALP) levels along with functional improvement criteria, which is, Medical research council (MRC) scale and North Star Ambulatory assessment (NSAA), assessed at baseline and following the intervention. Results: After the intervention, the SC levels significantly decreased from a mean baseline value of 9.28 mg/dL to 8.31 mg/dL (p-value = 0.02). With a p-value of 0.29, the mean CPK value dropped from 2192.33 IU/L to 1567.5 IU/L. Following the intervention, the ALP levels dropped from 200.33 to 75.5 U/L (p-value = 0.15). MRC scale improved in three out of six patients. NSAA remained stable. There were no adverse effects. Conclusion: This study has proven the safety of Neu REFIX beta-glucan food supplement and its efficacy in improving both plasma biomarkers and functional parameters of muscle in a short duration of 2 months. Further validation by evaluation of muscle function for a longer duration is recommended to confirm the efficacy of Neu-REFIX food supplement as a potential adjuvant DMT in muscular dystrophies.

Hepatoprotective Effects of Aureobasidium pullulans Derived ß 1,3-1,6 Glucans in a Murine Model of Non-alcoholic Steatohepatitis.

Background: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent conditions characterized by inflammation and fibrosis of the liver, which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Conventional modalities are mainly symptomatic, with no definite solution. Beta-glucan-based biological response modifiers are a potential strategy in lieu of their beneficial metabolic effects. Aureobasidium pullulans strains AFO-202 and N-163 beta-glucans were evaluated for anti-fibrotic and anti-inflammatory hepatoprotective potentials in a NASH animal model in this study. Methods: In the STAM™ murine model of NASH, five groups were studied for 8 weeks: (1) vehicle (RO water), (2) AFO-202 beta-glucan; (3) N-163 beta-glucan, (4) AFO-202+N-163 beta-glucan, and (5) telmisartan (standard pharmacological intervention). Evaluation of biochemical parameters in plasma and hepatic histology including Sirius red staining and F4/80 immunostaining were performed. Results: AFO-202 beta-glucan significantly decreased inflammation-associated hepatic cell ballooning and steatosis. N-163 beta-glucan decreased fibrosis and inflammation significantly (P value < 0.05). The combination of AFO-202 with N-163 significantly decreased the NAFLD Activity Score (NAS) compared with other groups. Conclusion: This preclinical study supports the potential of N-163 and AFO-202 beta-glucans alone or in combination as potential preventive and therapeutic agent(s), for NASH.

Two unique biological response-modifier glucans beneficially regulating gut microbiota and faecal metabolome in a non-alcoholic steatohepatitis animal model, with potential applications in human health and disease.

OBJECTIVE: The gut microbiome and its metabolites are influenced by age and stress and reflect the metabolism and health of the immune system. We assessed the gut microbiota and faecal metabolome in a static animal model of non-alcoholic steatohepatitis (NASH). DESIGN: This model was subjected to the following treatments: reverse osmosis water, AFO-202, N-163, AFO-202+N-163 and telmisartan treatment. Faecal samples were collected at 6 and 9 weeks of age. The gut microbiome was analysed using 16S ribosomal RNA sequences acquired by next-generation sequencing, and the faecal metabolome was analysed using gas chromatography-mass spectrometry. RESULTS: Gut microbial diversity increased greatly in the AFO-202+N-163 group. Postintervention, the abundance of Firmicutes decreased, whereas that of Bacteroides increased and was the highest in the AFO-202+N-163 group. The decrease in the abundance of Enterobacteriaceae and other Firmicutes and the abundance of Turicibacter and Bilophila were the highest in the AFO-202 and N-163 groups, respectively. Lactobacillus abundance was highest in the AFO-202+N-163 group. The faecal metabolite spermidine, which is beneficial against inflammation and NASH, was significantly decreased (p=0.012) in the N-163 group. Succinic acid, which is beneficial in neurodevelopmental and neurodegenerative diseases, was increased in the AFO-202 group (p=0.06). The decrease in fructose was the highest in the N-163 group (p=0.0007). Isoleucine and Leucine decreased with statistical significance (p=0.004 and 0.012, respectively), and tryptophan also decreased (p=0.99), whereas ornithine, which is beneficial against chronic immune-metabolic-inflammatory pathologies, increased in the AFO-202+N-163 group. CONCLUSION: AFO-202 treatment in mice is beneficial against neurodevelopmental and neurodegenerative diseases, and has prophylactic potential against metabolic conditions. N-163 treatment exerts anti-inflammatory effects against organ fibrosis and neuroinflammation. In combination, these compounds exhibit anticancer activity.

Beneficial Immune Regulation by Biological Response Modifier Glucans in COVID-19 and Their Envisaged Potentials in the Management of Sepsis.

Sepsis is a life-threatening condition caused by an abnormal immune response induced by infection with no approved or specific therapeutic options. We present our perspectives for the therapeutic management of sepsis through a four-way approach: (1) infection control through immune enhancement; (2) immune suppression during the initial hyper-inflammatory phase; (3) balanced immune-modulation to counter the later immune-paralysis phase; and (4) advantageous effects on metabolic and coagulation parameters throughout. COVID-19 is a virus-triggered, accelerated sepsis-like reaction that is associated with the rapid progress of an inflammatory cascade involving a cytokine storm and multiorgan failure. Here, we discuss the potential of the biological response modifiers, ß-glucans (BRMGs), in the management of sepsis based on their beneficial effects on inflammatory-immune events in COVID-19 clinical studies. In COVID-19 patients, apart from metabolic regulation, BRMGs, derived from a black yeast, Aureobasidium pullulans strain AFO-202, have been reported to stimulate immune responses. BRMGs, produced by another strain (N-163) of A. pullulans, have been implicated in the beneficial regulation of inflammatory markers and immunity, namely IL-6, C-reactive protein (CRP), D-Dimer, ferritin, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-C-reactive protein ratio (LCR), leucocyte-to-C-reactive protein ratio (LeCR), and leukocyte-to-IL-6 ratio (LeIR). Agents such as these ß-glucans, which are safe as they have been widely consumed by humans for decades, have potential as adjuncts for the prevention and management of sepsis as they exert their beneficial effects across the spectrum of processes and factors involved in sepsis pathology, including, but not limited to, metabolism, infection, inflammation, immune modulation, immune enhancement, and gut microbiota.

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

BACKGROUND: Chondrocytes 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. METHODS: In 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. RESULTS: The 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. CONCLUSION: This 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.

ß­glucan vaccine adjuvant approach for cancer treatment through immune enhancement (B­VACCIEN) in specific immunocompromised populations (Review).

The incidence of cancer, which is the second leading cause of mortality globally, continues to increase, although continued efforts are being made to identify effective treatments with fewer side­effects. Previous studies have reported that chronic microinflammation, which occurs in diseases, including diabetes, along with weakened immune systems, may ultimately lead to cancer development. Chemotherapy, radiotherapy and surgery are the mainstream approaches to treatment; however, they all lead to immune system weakness, which in turn increases the metastatic spread. The aim of the present review was to provide evidence of a biological response modifier ß­glucan [ß­glucan vaccine adjuvant approach to treating cancer via immune enhancement (B­VACCIEN)] and its beneficial effects, including vaccine­adjuvant potential, balancing metabolic parameters (including blood glucose and lipid levels), increasing peripheral blood cell cytotoxicity against cancer and alleviating chemotherapy side effects in animal models. This suggests its value as a potential strategy to provide long­term prophylaxis in immunocompromised individuals or genetically prone to cancer.

Beneficial effects of novel aureobasidium pullulans strains produced beta-1,3-1,6 glucans on interleukin-6 and D-dimer levels in COVID-19 patients; results of a randomized multiple-arm pilot clinical study.

OBJECTIVE: In this pilot clinical study, we report the beneficial effects of beta glucans derived from two strains AFO-202 and N-163 of a black yeast Aureobasidium pullulans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients. METHODS: A total of 24 RT-PCR positive COVID-19 patients were recruited and randomly divided into three groups (Gr): Gr. 1 control (n = 8) - Standard treatment; Gr. 2: Standard treatment + AFO-202 beta glucan (n = 8); and Gr. 3, Standard treatment + combination of AFO-202 and N-163 beta glucans (n = 8) for 30 days. RESULTS: There was no mortality or requirement of ventilation of the subjects in any of the groups. There was a decrease in D-Dimer values (751 ng/ml to 143.89 ng/ml) and IL-6 values (7.395-3.16 pg/ml) in Gr. 1 in 15 days but the levels increased to abnormal levels on day 30 (D-Dimer: 202.5 ng/ml; IL-6 55.37 pg/ml); which steadily decreased up to day 30 in groups 2 (D-dimer: 560.99 ng/dl to 79.615; IL-6: 26.18-3.41 pg/ml) and 3 (D-dimer: 1614 ng/dl to 164.25 ng/dl; IL-6: 6.25-0.5 pg/ml). The same trend was observed with ESR. LCR and LeCR increased while NLR decreased significantly in Gr. 3. CD4 + and CD8 + T cell count showed relatively higher increase in Gr.3. There was no difference in CRP within the groups. CONCLUSION: As these beta glucans are well known food supplements with a track record for safety, larger multi-centric clinical studies are recommended to validate their use as an adjunct in the management of COVID-19 and the ensuing long COVID-19 syndrome.

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-ß1, 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.

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-ßgal) 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-ßgal and started degenerating at 25 days. SA-ßgal 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.

AIMS: We 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 METHODS: OA-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 FINDINGS: The 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. SIGNIFICANCE: The 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 3D Polymer Scaffold Platform for Enhanced in vitro Culture of Human & Rabbit Buccal Epithelial Cells for Cell Therapies.

BACKGROUND: Buccal mucosal epithelial cells show promising application for various regenerative medicine approaches. In this study, we examined the feasibility of culturing rabbit and human buccal mucosal epithelial cells in a novel thermoreversible gelation polymer (TGP) scaffold, without feeder layers or other foreign proteins. METHODS & RESULTS: The results of this 28-day in vitro culture, u sing the conventional technique (2D) and TGP (3D) showed that the epithelial cell morphology could be maintained only in the TGP group while cells in the 2D group de-differentiated to fibroblast morphology in both human and rabbit samples. CK3 expression, a marker for epithelial differentiation was higher in 3D-TGP cultured cells than 2D. CONCLUSION: TGP based in vitro cell culture is a prospective methodology to culture buccal mucosal epithelial cells efficiently without using foreign biological components for tissue engineering applications.

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.

PURPOSE: McCarey-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. METHODS: Thirteen 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. RESULTS: MK + 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. CONCLUSION: TGP 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.

BACKGROUND: Chondrocyte 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. METHODS: Chondrocytes 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. RESULTS: In 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. CONCLUSION: The 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.