Amount Of Viable Cells Research Articles

Preparation of a Lactobacillus rhamnosus ATCC 7469 microencapsulated-lactulose synbiotic and its effect on equol production.

Equol is a highly active product of soy isoflavones produced by specific bacteria in the human or animal colon. However, equol production is influenced by differences in the gut flora carried by the body. Our previous research has shown that a synbiotic preparation comprising the probiotic Lactobacillus rhamnosus ATCC 7469 and the prebiotic lactulose can enhance equol production by modulating the intestinal flora. Nevertheless, the harsh environment of the gastrointestinal tract limits this capability by diminishing the number of probiotics reaching the colon. Microencapsulation of probiotics is an effective strategy to enhance their viability. In this study, probiotic gel microspheres (SA-S-CS) were prepared using an extrusion method, with sodium alginate (SA) and chitosan (CS) serving as the encapsulating materials. Scanning electron microscopy (SEM) was employed to observe the surface morphology and the internal distribution of bacteria within the microcapsules. The structural characteristics of the microcapsules were investigated using Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Furthermore, the thermal stability, storage stability, probiotic viability post-simulated gastrointestinal fluid treatment, and colon release rate were examined. Finally, the impact of probiotic microencapsulation on promoting equol production by the synbiotic preparation was assessed. The results indicated that the microcapsules exhibited a spherical structure with bacteria evenly distributed on the inner surface. Studies on thermal and storage stability showed that the number of viable cells in the probiotic microcapsule group significantly increased compared to the free probiotic group. Gastrointestinal tolerance studies revealed that after in vitro simulated gastrointestinal digestion, the amount of viable cells in the microcapsules was 7 log10 CFU g-1, demonstrating good gastrointestinal tolerance. Moreover, after incubation in simulated colonic fluid for 150 min, the release rate of probiotics reached 93.13%. This suggests that chitosan-coated sodium alginate microcapsules can shield Lactobacillus rhamnosus ATCC 7469 from the gastrointestinal environment, offering a novel model for synbiotic preparation to enhance equol production.

INFLUENCE OF HYDRODYNAMIC PARAMETERS ON THE SYNTHESIS OF TARGET METABOLITES AND THE DEGREE OF DISINTEGRATION DURING THE SUBMERGED CULTIVATION OF MICROMYCETES

One of the limiting parameters of the mycelial fungi submerged cultivation for the synthesis of metabolites used for medical, food and industrial purposes is the hydrodynamic conditions of submerged cultivation. The effect of mechanical stirring rate on the yield of metabolites was investigated on the example of cultivation of mycelial fungi Aspergillus awamori – glucoamylase producer. It was found that the maximum yield of the enzyme was observed at the stirring speed of turbine impeller amounting to 60-120 rpm. As the stirring speed increased, the amount of synthesized metabolite decreased, which is a consequence of the destruction of the mycelial cells. The connection between the decrease in glucoamylase synthesis and the rejuvenation of the culture caused by its recovery from fragments of the destroyed mycelium and the subsequent increase in the amount of biomass was shown. At the same time, the degree of destruction was shown by the increase in the amount of nucleic acids in the filtrate. The relationship between the degree of destruction of mycelial cells, determined by the amount of nucleic acids in the filtrate under extreme conditions of ultrasonic disintegration, and the viability of the culture was also demonstrated. The amount of viable cells increased at first (until the saturation of the culture fluid with nucleic acids), and then began to gradually decrease. The obtained results can serve as a starting point for further studies of the possibility of stimulating the primary accumulation of biomass of micromycetes by ultrasound.

Comparison of Harvesting and Processing Technique for Adipose Tissue Graft: Evaluation of Cell Viability

Background: Clinical studies demonstrated the efficacy of therapies based on the autologous grafting of adult mesenchymal stem cells to accelerate the healing and regenerative processes of the skin and mesenchymal tissues; therefore, it is considered a valuable approach in the aesthetic rejuvenation treatment to give volumization and skin regeneration effects. Objective: The aim of the project consisted of the control of cell viability of adipose tissue (AT) harvested using the two types of cannulas having 0.8 mm and 1 mm side port holes. The results were compared with tissue harvested with a standard liposuction technique and processed with a standard procedure consisting of enzymatic digestion (collagenase). Methods: This study was performed on adipose tissues harvested from 7 patients (6 females and 1 male) with an average age of 48.5 years with 3 different techniques. We compared the cell vitality of every sample at T0 and T72. Results: Lipoaspirate tissue-derived by 0.8- and 1 mm cannula from all samples proved to be vital and possess viable cells. The average absorbance was similar immediately after plating (T0) and 72 hours after (T72) for the two cannulas, 0.8- and 1 mm cannula. The two systems proved to equally harvest vital tissue. An increase in cell viability was observed in all samples for each condition (0.8-, 1 mm and enzymatic digestion). Conclusion: This study proved that guided harvested adipose tissue with small cannulas with small side port holes yields a comparable amount of viable cells compared to adipose tissue harvested with a liposuction system and processed with enzymatic digestion (collagenase). This study confirms that the minimally invasive technique and minimal manipulation of the adipose tissue could yield a tissue with a good amount of viable cells. This micro fragmented adipose tissue is a promising source for regenerative treatments.

The effect of different vitrification protocols on cell survival in human ovarian tissue: a pilot study

BackgroundVitrification has superseded the slow freezing method for cryopreservation of oocytes, embryos, and sperm, but there are as yet no standard protocols for its use in ovarian tissue cryopreservation (OTC). Published protocols diverge mainly with regard to the extent of supplementation of dimethyl sulfoxide (DMSO) to the vitrification medium, and to the use of an open or closed vitrification system.We investigated the viability of cells after vitrification/warming, using ovarian tissue of transgender patients, by means of Fluorescence Activated Cells Sorting (FACS), and histomorphological analyses using a DMSO-containing (P1) and a DMSO-free protocol (P2) in an open or closed vitrification setting.ResultsTwelve ovarian samples were donated from female-to-male transgender patients: 6 were vitrified according to protocol 1, the other 6 according to protocol 2. The amount of viable cells was 90.1% (P1) and 88.4% (P2) before vitrification. After vitrification and subsequent warming, viable cells were reduced to 82.9% (P1, p = 0.093) and 72.4% (P2, p = 0.019). When comparing the closed and the open systems, the decline in cell viability from pre- to post-vitrification was significant only for the latter (p = 0.037). Histological examination reveals no significant differences with respect to degenerated follicles before or after vitrification.ConclusionThese results led us to conclude that a protocol containing DMSO results in a higher viability of ovarian cells than a protocol that uses ethylene glycol as cryoprotective agent in vitrification. The use of an open vitrification system led to significant decline in the rate of viable cells.Trial registrationNCT03649087, retrospectively registered 28.08.2018.

P–131 Significance of the phenomenon of blastomere exclusion from compaction: Its relation to irregular cleavage, blastocyst development rate, and pregnancy rate

Abstract Study question When does blastomere exclusion from compaction increase and what effect does it have on the embryo? Summary answer More blastomere were excluded from compaction in embryos with irregular cleavage, resulting in lower blastocyst development rates, but no decrease in pregnancy rates at transfer. What is known already It has been reported that many of the chromosome analysis results of blastomere excluded from compaction were aneuploid, and pointed out that this exclusion may be related to the repair of blastocyst euploidy, but the effect of the number of excluded blastomere has not been reported. Study design, size, duration This is a retrospective study of 578 embryos that developed into morula with time-lapse monitoring by EmbryoScope (Vitrolife) in 2018–2019. Participants/materials, setting, methods The target embryos were classified into two groups: embryos with normal first and second cleavage (normal cleavage group) and embryos with irregular cleavage (dynamics of one cell dividing into three or more cells), called “direct cleavage”, at either cleavage (DC group), and the number of blastomere excluded from compaction during morula formation was recorded and compared. The blastocyst development rate and single blastocyst transfer pregnancy rates of the two groups were compared. Main results and the role of chance There are 286 in the normal cleavage group and 292 in the DC group. The mean number of excluded blastomere was 0.76 and 3.55, respectively, which was significantly higher in the DC group (P < 0.01). Good blastocyst (Gardner classification 4 or higher) development rate was 84.5% (239/283) and 65.8% (181/275), respectively, and high grade blastocyst (Gardner classification BB or higher) development rate was 43.9% (105/239) and 14.9% (27/181) of them, both significantly higher in the normal cleavage group (P < 0.01). The single blastocyst transfer pregnancy rates were 31.6% (25/79) and 32.4% (11/34), and the miscarriage rates were 24.0% (6/25) and 27.3% (3/11), respectively, neither was there a significant difference between the two groups. So, direct cleavage increased the number of blastomere excluded from compaction, decreased the rate of morula to good blastocyst development and reduced blastocyst grade, but did not affect blastocyst transfer pregnancy rate and miscarriage rate. Limitations, reasons for caution Please note that all target embryos must have developed into morula or larger (embryos that did not develop into morula will not be included in the study). Wider implications of the findings: Severe chromosomal aberrant blastomeres formed by direct cleavage were excluded from compaction, and the blastocyst development rate decreased due to a decrease in the amount of viable cells, but it is suggested that this blastomere exclusion mechanism is not related to euploidy after blastocyst development. Trial registration number Not applicable

The prokaryotic community in an extreme Antarctic environment: the brines of Boulder Clay lakes (Northern Victoria Land)

During summer 2014, three hypersaline brines were discovered in two frozen lakes of Boulder Clay (Northern Victoria Valley, Antarctica). Ongoing research seeks to gain novel insights on the microbial ecology of such environments, in order to further the understanding of life adaptation to extreme conditions. To this aim, the abundance of prokaryotic cells (including cell morphologies and size for biomass conversion), the amount of viable cells (in terms of membrane-intact cells and respiring cells), the viral count, the physiological profiles at community level and the main microbial enzymatic activities were described. The brines differed each other in terms of prokaryotic cells’ abundance, size, and viability as well as viral abundance. Cell morphotypes and metabolic responses also varied among the brine samples. Underground interconnections were likely to occur, with the microbial community becoming more abundant and structured to better exploit the limited resource availability. Overall, complex interactions among multiple environmental factors, including marine water origin, depth horizon, isolation time of the brines, and climatic variations, reflected on the microbial community distribution patterns and highlighted the need to preserve these niches of extreme life.

Comparing the efficacy of γ- and electron-irradiation of PBMCs to promote secretion of paracrine, regenerative factors

Cell-free secretomes represent a promising new therapeutic avenue in regenerative medicine, and γ-irradiation of human peripheral blood mononuclear cells (PBMCs) has been shown to promote the release of paracrine factors with high regenerative potential. Recently, the use of alternative irradiation sources, such as artificially generated β- or electron-irradiation, is encouraged by authorities. Since the effect of the less hazardous electron-radiation on the production and functions of paracrine factors has not been tested so far, we compared the effects of γ- and electron-irradiation on PBMCs and determined the efficacy of both radiation sources for producing regenerative secretomes. Exposure to 60 Gy γ-rays from a radioactive nuclide and 60 Gy electron-irradiation provided by a linear accelerator comparably induced cell death and DNA damage. The transcriptional landscapes of PBMCs exposed to either radiation source shared a high degree of similarity. Secretion patterns of proteins, lipids, and extracellular vesicles displayed similar profiles after γ- and electron-irradiation. Lastly, we detected comparable biological activities in functional assays reflecting the regenerative potential of the secretomes. Taken together, we were able to demonstrate that electron-irradiation is an effective, alternative radiation source for producing therapeutic, cell-free secretomes. Our study paves the way for future clinical trials employing secretomes generated with electron-irradiation in tissue-regenerative medicine.

Rapid detection of antimicrobial resistance markers with Allplex™ Entero-DR assay directly from positive blood culture bottles.

A method for rapid detection of one extended-spectrum β-lactamase (ESBL) and five carbapenemase-encoding genes as well as vancomycin resistance markers directly from blood cultures using the Allplex™ Entero-DR assay (Seegene, Seoul, South Korea) is presented. Altogether 28 previously well-characterized resistant Gram-negative bacilli and Enterococcus spp., and 142 clinical blood cultures containing Gram-negative bacilli or Gram-positive cocci were analyzed. The method had 100% sensitivity and specificity for detecting blaOXA-48-like, blaKPC, blaVIM, blaIMP, blaNDM, blaCTX-M, vanA, and vanB. The lowest detectable amount of viable cells in blood culture samples were 5.39·104 CFU/mL, 6.66·104 CFU/mL, 5.13·103 CFU/mL, 6.09·104 CFU/mL, 6.66·104 CFU/mL, 6.66·104 CFU/mL, 3.12·104 CFU/mL, and 5.34·104 CFU/mL for the blaKPC, blaOXA-48-like, blaVIM, blaIMP, blaNDM, blaCTX-M, vanA, and vanB, respectively. The results were available within 90 min from signal positive blood cultures, as no separate DNA extraction steps were needed, and the assay showed no interference from blood or culture media used allowing reliable and simplified detection of the resistance markers.

Optimization of the status of cell growth guided by an on-line biomass sensor for polymalic acid fermentation

Abstract Polymalic acid (PMA) is a water-soluble biopolymer, composed of the monomer malic acid (MA), produced by Auerobasidium pullulans. In this study, an on-line biomass monitor was used to evaluate the status of viable biomass from seed culture to fermentation for PMA production. The amount of viable cells was sensitive to regulation by available nitrogen. From the exponential phase, cell morphology became looser and larger as demonstrated by the fc value and light microscopy. These findings were consistent with the average cell circumference, which increased from 10.8 ± 1.31 to 12.4 ± 1.90 μm. Under the optimized seed culture, a comparable fed-batch strategy was developed to balance cell growth and PMA biosynthesis in a 5-L fermentor, achieving PMA titers up to 128.3 ± 1.51 g/L with a corresponding yield of 0.54 g/g glucose. The process was further guided by the biomass sensor and scaled-up in a 50-L fermentor, producing 144.3 ± 1.8 g/L PMA (or 163.8 g/L malic acid after hydrolysis) with a maximum MA productivity and yield of 0.91 g/L h and 0.60 g/g, respectively. This study will helpful for understanding the relationship between cell growth and PMA biosynthesis and provided an operable fermentation process for large-scale PMA production.

Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix

Bacterial biofilms play a key role during infections, which are associated with an increased morbidity and mortality. The classical antibiotic therapy cannot eradicate biofilm-related infections because biofilm bacteria display high drug resistance due to biofilm matrix. Thus, novel drug delivery to overcome biofilm resistance and eliminate biofilm-protected bacteria is needed to be developed. In this study, positively charged chitosan nanoparticles (CSNP) loaded with oxacillin (Oxa) and Deoxyribonuclease I (CSNP-DNase-Oxa) were fabricated. The antibiofilm activity was evaluated against Staphylococcus aureus biofilms. Biofilm architecture on silicone surfaces was investigated by scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) was used to examine live/dead organisms within biofilm. CSNP-DNase-Oxa exhibited higher antibiofilm activity than Oxa-loaded nanoparticles without DNase (CSNP-Oxa) and free Oxa (Oxa and Oxa + DNase) at each concentration in all in-vitro tests. CSNP-DNase-Oxa inhibited biofilm formation in-vitro and eradicated mature biofilm effectively. CSNP-DNase-Oxa could disrupt the biofilm formation through degradation of eDNA, reduced biofilm thickness and the amount of viable cells on silicone. Repeated treatment with CSNP-DNase-Oxa for two days resulted in 98.4% biofilm reduction. Moreover, CSNP-DNase-Oxa was not only able to affect the biofilm of a standard S. aureus strain, but also showed the highest eradication of biofilms of clinical isolates compared with control groups. These results suggest the potential applicability of NPs for the treatment of biofilm-related infections and provide a platform for designing novel drug delivery with more functions.

Enterococcus hirae biofilm formation on hospital material surfaces and effect of new biocides

BackgroundNowadays, the bacterial contamination in the hospital environment is of particular concern because the hospital-acquired infections (HAIs), also known as nosocomial infections, are responsible for significant morbidity and mortality. This work evaluated the capability of Enterococcus hirae to form biofilm on different surfaces and the action of two biocides on the produced biofilms.MethodsThe biofilm formation of E. hirae ATCC 10541 was studied on polystyrene and stainless steel surfaces through the biomass quantification and the cell viability at 20 and 37 °C. The effect of LH IDROXI FAST and LH ENZYCLEAN SPRAY biocides on biomasses was expressed as percentage of biofilm reduction. E. hirae at 20 and 37 °C produced more biofilm on the stainless steel in respect to the polystyrene surface. The amount of viable cells was greater at 20 °C than with 37 °C on the two analyzed surfaces. Biocides revealed a good anti-biofilm activity with the most effect for LH ENZYCLEAN SPRAY on polystyrene and stainless steel at 37 °C with a maximum biofilm reduction of 85.72 and 86.37%, respectively.ResultsE. hirae is a moderate biofilm producer depending on surface material and temperature, and the analyzed biocides express a remarkable antibiofilm action.ConclusionThe capability of E. hirae to form biofilm can be associated with its increasing incidence in hospital-acquired infections, and the adoption of suitable disinfectants is strongly recommended.

Chitosan-dextran sulfate hydrogels as a potential carrier for probiotics

Physical and chemical (crosslinked with genipin) hydrogels based on chitosan and dextran sulfate were developed and characterized as novel bio-materials suitable for probiotic encapsulation. The swelling of the hydrogels was dependent on the composition and weakly influenced by the pH of the media. The morphology analysis supports the swelling data showing distinct changes in microstructure depending on the composition. The viability and culturability tests showed approx. 3.6 log CFU/mL decrease of cells (L. acidophilus as model) incorporated into chemical hydrogels when compared to the number of viable native cells. However, the live/dead viability assay evidenced that a considerable amount of viable cells were still entrapped in the hydrogel network and therefore the viability is most likely underestimated. Overall, the developed systems are robust and their structure, rheology and swelling properties can be tuned by changing the blend ratio, thus constituting appealing bio-matrices for cell encapsulation.

Isolation and characterization of cardiac progenitor cells from myocardial right atrial appendage tissue

Resident stem cells of the heart are denoted as heterogeneous population of immature cells, which reside in the myocardium and characterized by their ability to self-renewal and are multipotent differentiation capacity into cardiomyocyte-like and vascular like cells. CSCs were originally isolated directly by long enzymatic digestion of heart tissue and selection using stem cell markers. However, long exposure to enzymatic digestion and small myocardial sample size can affect the possibility of obtaining a significant amount of viable cells. To avoid these problems, we developed a method consisting of growing of the CPC in explant culture and subsequent immunomagnetic selection.

Abstract 2063: Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells

Abstract Aim of the study was to prepare and characterize RGD grafted PEGylated liposomes of gemcitabine (PLGs) and to evaluate its cellular uptake, in vitro anti-proliferative activity and apoptotic effect in siRNA pretreated lung cancer cells. PLGs were prepared by thin film hydration method and optimized for particle size, zeta potential and entrapment efficiency. Functionalization of liposomes was done by coupling reaction between DSPE mPEG and cRGD peptide by maleimide based reaction. Similarly, RGD grafted RRM1 siRNA liposomes were also formulated and evaluated. MTT assay was done to determine IC50 values of RGD grafted PLGs in A549 and H1299 cancer cells which were pre-treated with RGD grafted RRM1 siRNA loaded liposomes at a concentration of 50 pM of siRNA for gene silencing. DNA content analysis was done by flow cytometry using rhodamine in A549. The mode of cell death at different time and concentration was determined by FITC-AnnexineV assay in A549 cells and confocal microscopy was performed to assess the potential of RGD grafting on cellular uptake. RGD conjugated and unconjugated PLGs were found Nano sized and had negative zeta potential with entrapment of 65%. H1299 cell line showed more amount of viable cells after 48 hr as compared to A549 cells in RGD conjugated PLGs. siRNA pre-treated PLGs exposed cells showed significantly less IC50 values as compared to cells without siRNA pretreatment and non-grafted liposomes. The results showed that the RGD conjugated liposomes at the concentration of 7nM showed 46% G1 phase arrest in siRNA pretreated cells as compared to 22% G1 phase arrest without prior siRNA treatment at 16hr. Two types of mode of cell death were found during the FITC-Annexine V assay. At 24 hr, the treatment with RGD grafted PLGs resulted in 17% & 4.4% necrotic & apoptotic cell death respectively. While at equivalent drug concentration, the PLGs and drug solution showed 5.3% & 32.2% and 4.2% & 29.6% necrotic & apoptotic cell death respectively. Furthermore, the apoptosis was found to be time and concentration dependent. Results substantiate the sensitization effect by pre-exposure of siRNA in liposomal forms at Pico molar concentration along-with phagocytosis as mechanism of uptake of RGD-grafted liposomes. To conclude, prior silencing of the resistance imparting gene can manifest the effect of therapy by conferring improved sensitivity in cancer cell lines. The effect can further be augmented by employing receptor targeting peptides such as RGD. Hence, Nano-constructs of chemotherapeutic drugs conjugated with RGD can effectively target lung cancer cells and pretreatment of RRM1 siRNA can probably reduce the limitation of drug resistance associated with lung cancer chemotherapy. Citation Format: Rohan A. Lalani, Priyanka Bhatt, Mohan Rathi, Ambikanandan Misra. Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2063.

Immunogenic cell death by oncolytic herpes simplex virus type 1 in squamous cell carcinoma cells.

Molecules essential for the induction of immunogenic cell death (ICD) are called damage-associated molecular patterns (DAMPs). The effects of oncolytic herpes simplex virus type 1 (HSV-1) on the production of DAMPs were examined in squamous cell carcinoma (SCC) cells. The cytopathic effects of HSV-1 RH2 were observed in mouse SCCVII cells infected at a high multiplicity of infection (MOI), and the amounts of viable cells were decreased. After being infected with RH2, ATP and high mobility group box 1 (HMGB1) were released extracellulary, while calreticulin (CRT) translocated to the cell membrane. A flow-cytometric analysis revealed an increase in the number of annexin-V and propidium iodide (PI)-stained cells; and the amount of cleaved poly (ADP-ribose) polymerase (PARP) was increased. The killing effect of RH2 was reduced by pan-caspase inhibitor z-VAD-fmk and the caspase-1 inhibitor z-YVAD-fmk, suggesting the involvement of apoptosis and pyroptosis. In C3H mice bearing synergic SCCVII tumors, the growth of tumors injected with the supernatant of RH2-infected cells was less than that of tumors injected with phosphate-buffered saline (PBS). These results indicate that oncolytic HSV-1 RH2 produces DAMPs from SCC cells to induce cell death. This may contribute to the enhancement of tumor immunity by oncolytic HSV-1.

Mo1707 Serial Treatment With Anti-TNF Ab Reduces Tumor Burden in a Murine Model of Chronic Colitis, Compared to Single Dose Anti-TNF

Background: Local application of adipose-derived mesenchymal stem cells (ADSCs) is a promising method for the treatment of perianal fistula in patients with Crohn's disease. There is a lack of data evaluating the distribution and viability of ADSCs after application. Therefore more studies are needed including the technique of in vivo monitoring. Aim: The aim of the study was to evaluate the effect of ADSCs application on fistula healing regarding their viability and distribution in perifistular region. Methods: Coecostomy was used as a fistula model in Lewis rats. The inguinal adipose tissue was harvested from transgenic donor expressing firefly luciferase (LEW-Tg(Rosa-luc)11Jmsk; Jichi Medical School, Japan). The ADSCs were isolated using collagenase technique and injected (1-2*106 cells/ml) in perifistular tissue of 16 rats. After application of D-luciferin (25mg/kg), animals were imaged in IVIS Lumina XR camera on days 0, 2, 7, 14 and 30. Fistula drainage assessment (FDA) was used to evaluate the fistula healing. Animals from interventional group were compared to 30 rats with untreated fistula. Results: There was no morbidity or mortality after intervention. The fistula was identified as healed according to FDA in 6 (37.5%) animals after ADSCs injection vs. 1 case (3.3%) with spontaneous closure in control group (p< 0.0047). Bioluminescence was strongest 2 days after application (31.2*104 (6.10-11.0*104). The amount of viable cells remained stable during first 7 days, which confirms successful cells seating. The signal started to decrease slowly afterwards day 14: 6.4*104 (0.5-28.8*104) and day 30: 2.8*104 (0.16-16.9*104) (values are medians in p/s/cm2/sr) (fig.1). The luminescence 30 days after injection was significantly higher in animals with healed fistula 8.23*104 (1.18*1041.70*105) vs. 2.75*104 (4.93*103-6.88*104); p=0.18. Conclusion: Perifistular application of ADSCs and in vivo monitoring using bioluminescence are technically feasible and doesn't cause mortality or morbidity. Local treatment with ADSCs led to significantly more frequent fistula closure compared to the control group. The fistula healing was associated with significantly higher concentration of viable ADSCs in perifistular region, which was confirmed by stronger bioluminescence signal. Supported by NT13708

Mo1706 Local Application of Adipose-Derived Mesenchymal Stem Cells Can Lead to Intestinal Fistula Healing - Experimental Study

Background: Local application of adipose-derived mesenchymal stem cells (ADSCs) is a promising method for the treatment of perianal fistula in patients with Crohn's disease. There is a lack of data evaluating the distribution and viability of ADSCs after application. Therefore more studies are needed including the technique of in vivo monitoring. Aim: The aim of the study was to evaluate the effect of ADSCs application on fistula healing regarding their viability and distribution in perifistular region. Methods: Coecostomy was used as a fistula model in Lewis rats. The inguinal adipose tissue was harvested from transgenic donor expressing firefly luciferase (LEW-Tg(Rosa-luc)11Jmsk; Jichi Medical School, Japan). The ADSCs were isolated using collagenase technique and injected (1-2*106 cells/ml) in perifistular tissue of 16 rats. After application of D-luciferin (25mg/kg), animals were imaged in IVIS Lumina XR camera on days 0, 2, 7, 14 and 30. Fistula drainage assessment (FDA) was used to evaluate the fistula healing. Animals from interventional group were compared to 30 rats with untreated fistula. Results: There was no morbidity or mortality after intervention. The fistula was identified as healed according to FDA in 6 (37.5%) animals after ADSCs injection vs. 1 case (3.3%) with spontaneous closure in control group (p< 0.0047). Bioluminescence was strongest 2 days after application (31.2*104 (6.10-11.0*104). The amount of viable cells remained stable during first 7 days, which confirms successful cells seating. The signal started to decrease slowly afterwards day 14: 6.4*104 (0.5-28.8*104) and day 30: 2.8*104 (0.16-16.9*104) (values are medians in p/s/cm2/sr) (fig.1). The luminescence 30 days after injection was significantly higher in animals with healed fistula 8.23*104 (1.18*1041.70*105) vs. 2.75*104 (4.93*103-6.88*104); p=0.18. Conclusion: Perifistular application of ADSCs and in vivo monitoring using bioluminescence are technically feasible and doesn't cause mortality or morbidity. Local treatment with ADSCs led to significantly more frequent fistula closure compared to the control group. The fistula healing was associated with significantly higher concentration of viable ADSCs in perifistular region, which was confirmed by stronger bioluminescence signal. Supported by NT13708

Loss of CD62L Expression in CD4+FoxP3+ Regulatory T Cells Following Freeze-and-Thaw Prevents Protection Against GVHD in Murine Models

Backgrounds: CD4+FoxP3+ regulatory T cells (Treg) have been shown in pre-clinical murine models and human clinical trials to protect against acute graft-versus-host (GVHD) disease following hematopoietic cell transplantation (HCT). In murine models, Treg expression of CD62L is required for in vivo protection of GVHD (Strober 2005/Blazer 2004). Certain aspects of Treg isolation and administration could affect CD62L expression, such as the use of G-CSF mobilization or freeze-and-thaw. We hypothesized that freeze-thaw of Tregs could reduce CD62L expression and prevent Tregs from protecting against GVHD. Furthermore, we hypothesized that the loss of CD62L expression is mediated at least in part by metalloproteinases (MP).Methods and Results: We used established protocols of GVHD, in which lethal dose of fractionated total body irradiation was followed by murine bone marrow transplantation across major histocompatibility barriers. We sort-purified CD4+Foxp3+ Tregs based on CD25+(high) expression and measured baseline expression of CD62L. Cells were then gradually frozen in fetal calf serum/10%DMSO, and finally stored in liquid nitrogen for at least 7 days. After thawing Tregs showed an average of 58% of the baseline CD62L expression (CD62L expression fresh vs. frozen p=0.0009, n=5). Binding of frozen Tregs to plate bound L-Selectin ligand Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in vitrowas significantly reduced as compared to fresh Tregs (p=0.0003). To test the potential effect of freeze and thaw in vivo, we used an established murine model in which Tregs are given prior to conventional T cells (Tcon) to protect against GHVD. At the time of transplant conditioned BALB/c donors received 5x10^6 whole bone marrow and 5x10^5 CD4+CD25(high) Tregs (day 0), followed by 1x10^6 Tcon (day +2). Frozen/thawed Tregs and fresh Tregs respectively were given at the same time point at the same quantity of viable cells. Using luciferase-expressing cells (luc+) and BLI imaging we could show that homing of frozen luc+Tregs into pLN at day+5 was impaired as compared to fresh Tregs (p=0.0005). Relative weight loss as an indicator for GVHD was significantly higher and survival was significantly reduced in mice receiving frozen Treg in comparison to mice receiving fresh Treg. To confirm the importance of CD62L expression on Treg function we blocked CD62L in vivo by incubating fresh Tregs with monoclonal blocking antibody Mel14 (120ug/ml) for 1 hour prior to injection. A significantly higher proliferation of luc+ Tcon was observed in mice injected with Mel14-treated Tregs as compared to isotype control-treated Tregs. To test the potential role of metalloproteinases in cleaving CD62L following freeze and thaw, we added various metalloproteinase inhibitors (MPI) during the freezing process. We found that the addition of at least one MPI GM6001 (50nM) can diminish the loss of CD62L.Conclusion: Augmenting or manipulating Tregs of the donor graft is a promising area of cellular therapy that could be used to prevent GVHD in HCT and in potentially many other clinical contexts. We have found that the process of freeze and thaw reduces CD62L expression on Tregs and reduces protection against GVHD. Further, MPs may play role in the loss of CD62L expression and the use of MPIs for frozen products might preserve Treg function. DisclosuresNo relevant conflicts of interest to declare.

Molecular Imaging of Apoptosis for Early Prediction of Therapy Efficiency

Evasion of apoptosis is one of the hallmarks of cancer and any effective therapy primarily attempts to induce apoptosis. The evaluation of the degree of success of cancer therapy is currently mainly based on clinical and laboratory parameters and in a later stage on tumor shrinkage. However, none of these parameters provide an objective and early analysis of a therapeutic effect. Molecular imaging may provide a tool for this purpose by using not only pathophysiological but also biochemical effects of the therapy. First in the field, FDG-PET has been explored and demonstrated to offer insight in the amount of viable cells, even though false positives are commonly due to the lack of specificity of this particular radiopharmaceutical. More specific markers target the dying cells instead of those remaining alive. Specific apoptosis markers have been developed of which the radiolabeled Annexin A5 is the most intensely studied probe. Site-specific labeling strategies have improved this imaging probe with good results both in pre-clinical studies and in clinical trials, with promises for clinical applications. Caspase sensitive probes, such as the isatines, can also effectively image apoptosis but are limited due to the high background activities. More recent discoveries of small apoptosis sensitive probes, such as (18)F-ML10, are currently being explored. In this review, the most important apoptosis sensitive probes are described from both a pre-clinical and a clinical perspective, highlighting their potential but also their limitations as an early marker for therapeutic success. It seems that apoptosis imaging can help to guide therapy, not by replacing the current methodology but by providing additional and useful information.

Isolation of Stem Cells of the Degenerated Lumbar Intervertebral Disc Using a Low Cost and Very Effective Technique

Introduction The limitations of regenerative therapy of the tissue using stem cells are, among others, the length of time until a satisfactory amount of viable cells is obtained for the therapy and the high financial cost. The purpose of this study, was to report a low cost and very effective technique to obtain large amounts of adult mesenchymal stem cells from the degenerated intervertebral disc in a short time. Materials and Methods The methodology of the isolation in this study gave rise to an intellectual property claim deposited at INPI under number BR 10 2013 031330 0. The intervertebral disc in this study was obtained from patients with lumbar disc degenerative disease refractory to conservative treatment, who required surgery as therapy. Isolation consisted of incubating the degenerated intervertebral disc without going through enzymatic or mechanical dissociation in DMEM medium supplemented with 10% SFB and 1% P/S in a humidified oven at 37°C for 3 days. To prove that these cells were adult mesenchymal stem cells, the supernatant cells were collected with a Pasteur pipette and cultured conventionally in culture containers adding growth factors (0.02 ng/µL of EGF and FGF) for another 3 days. For this purpose, cellular adhesion tests, with fibroblastic characteristics, of the presence/absence of specific surface antigens through flow cytometry (CD 105+, CD73+, CD90+, CD45-, CD34-, CD14-, CD11b-, CD19-, HLA-DR-) and of cell-differentiation for adipogenic and osteogenic tissue were performed. Results Using this process, a confluence of ∼100% of adult mesenchymal stem cells was found in the culture containers 3 days after the patient's intervertebral disc was removed. The isolated cells presented percentages of antigens with specific surfaces in flow cytometry that were positive for mesenchymal stem cells, could differentiate into adipogenic and osteogenic tissue, and had an adhesive characteristic with fibroblastic morphology. The greater the degeneration, the smaller was the percentage of growth and isolation of cells, however its differentiation was satisfactory for adipocytes and osteocytes. Conclusion The benefits of the technique described are that it allows: (1) obtaining quickly and (2) a large amount of adult mesenchymal cells, using a methodology (3) with a low financial cost. Disclosure of Interest A. Falavigna: Conflict with CNPq, FAPERGS, AOSpine Latin America M. Peletti-Figueiró: None declared I. Aguiar: None declared M. Roesch-Ely: None declared D. Machado: None declared J. A. Henriques: None declared