Validation of the Media Fill Method for Solid Tissue Processing in Good Manufacturing Practice-Compliant Cell Production.

Over the past few years, a large number of clinical studies for advanced therapy medicinal products have been registered and/or conducted for treating various diseases around the world and many have generated very exciting outcomes. Media fill, the validation of the aseptic manufacturing process, is the simulation of medicinal product manufacturing using nutrient media. The purpose of this study is to explain the media fill procedure stepwise in the context of cellular therapy medicinal products. The aseptic preparation of patient individual cellular product is simulated by using tryptic soy broth as the growth medium, and sterile vials as primary packaging materials.

Isolation, Culture, Cryopreservation, and Preparation of Umbilical Cord-Derived Mesenchymal Stem Cells as a Final Cellular Product Under Good Manufacturing Practices-Compliant Conditions.

Mesenchymal stem cells have gained popularity in cell-based therapies due to their regenerative capabilities, immunomodulation properties, and paracrine activity through trophic factors. It is of utmost importance to establish clinical-grade procedures for the preparation of the mesenchymal stem cells for clinical applications. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of mesenchymal stem cells derived from umbilical cord as a final product under good manufacturing practices-compliant conditions.

Isolation, Culture, Cryopreservation, and Preparation of Skin-Derived Fibroblasts as a Final Cellular Product Under Good Manufacturing Practice-Compliant Conditions.

Cell-based therapies have become a popular approach in the field of regenerative medicine. Human fibroblast cells, one of the cell types widely used in clinical applications, have been used for skin regeneration and wound healing procedures. Furthermore, they are utilized for aesthetic purposes since fibroblasts lose their abilities such as collagen synthesis with age. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of fibroblasts derived from adult human skin as a final product under good manufacturing practice-compliant conditions.

Determination of antibiotic impurities in good manufacturing practices-grade cell therapy medicinal products.

Backrounds: According to the regulations of the health autorities, cell-based therapy products must be manufactured in good manufacturing production (GMP) facilities, fulfilling the required GMP standards. Products developed under the high quality control (QC) necessarity need to be approved for some QC tests. One of the main residual test is antibiotic test and this test should be validated. The aim of this study is to validate and determine the methods of detection of the antibiotic residue in the final product.Methods: Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) methods were used for the main steps of the production procedure, as well as the final products. Pharmaceutical Grade penicillin G and streptomycin sulfate were used as positive controls.Results: The results suggest that penicillin is broken down during cell culture and streptomycin is eliminated at the first washing step of the final product manufacture. It is shown in this study that LC-MS/MS method is one of the convenient method to test residual anibiotics and can be used to detect the antibiotic residues in cellular therapy products.Discussion: Since the antibiotic residues are eliminated in the final product and also it could be suggested that the methodology we followed is sufficiently safe and final product is pure.

Efficacy of stem cell therapy in ambulatory and nonambulatory children with Duchenne muscular dystrophy - Phase I-II.

PURPOSE: Duchenne muscular dystrophy (DMD) is an X-linked recessive pediatric disorder that ultimately leads to progressive muscle degeneration. It has been known that cell-based therapies were used to promote muscle regeneration. The main purpose of this study was to investigate the effects of allogeneic Wharton jelly-derived mesenchymal stem cells therapy in Duchenne muscular dystrophy. PATIENTS AND METHODS: Four ambulatory and five nonambulatory male patients were assessed as having acceptance criteria. Gene expression and immunohistochemical analysis were performed for dystrophin gene expression. The fluorescent in situ hybridization method was used for detection of chimerism and donor-recipient compatibility. Complement dependent lymphocytotoxic crossmatch test and detection of panel reactive antigen were performed. All patients were treated with 2 × 106 cells/kg dose of allogeneic Wharton jelly-derived mesenchymal stem cells via intra-arterial and intramuscular administration. Stability was maintained in patient follow-up tests, which are respiratory capacity tests, cardiac measurements, and muscle strength tests. RESULTS: The vastus intermedius muscle was observed in one patient with MRI. Chimerism was detected by fluorescent in situ hybridization and mean gene expression was increased to 3.3-fold. An increase in muscle strength measurements and pulmonary function tests was detected. Additionally, we observed two of nine patients with positive panel reactive antigen result. CONCLUSION: All our procedures are well tolerated, and we have not seen any application-related complications so far. Our main purpose of this study was to investigate the effects of allogeneic mesenchymal stem cell therapy and determine its suitability and safety as a form of treatment in this untreatable disorder.

A new approach for development of vaccine against visceral leishmaniasis: Lipophosphoglycan and polyacrylic acid conjugates.

OBJECTIVE: To determine the antileishmanial vaccine effectiveness of lipophosphoglycan (LPG) and polyacrylic acids (PAA) conjugates on in vivo mice models. METHODS: LPG molecule was isolated and purified from large-scale Leishmania donovani parasite culture. Protection efficacies of LPG alone, in combination with Freund's adjuvant, in a physical mixture and in conjugate (consisting of various LPG concentrations) with PAA, were comparatively determined by various techniques, such as cultivation with the micro-culture method, assessment of in vitro infection rates of peritoneal macrophages, determination of parasite load in liver with Leishman-Donovan Units, and detection of cytokine responses. RESULTS: Obtained results demonstrated that the highest vaccine-mediated immune protection was provided by LPG-PAA conjugate due to all parameters investigated. According to the Leishman-Donovan Units results, the sharpest decline in parasite load was seen with a ratio of 81.17% when 35 µg LPG containing conjugate was applied. This value was 44.93% for the control group immunized only with LPG. Moreover, decreases in parasite load were 53.37%, 55.2% and 65.8% for the groups immunized with 10 µg LPG containing LPG-PAA conjugate, a physical mixture of the LPG-PAA, and a mixture of LPG + Freund's adjuvant, respectively. Furthermore, cytokine results supported that Th1 mediated protection occurred when mice were immunized with LPG-PAA conjugate. CONCLUSIONS: It has been demonstrated in this study that conjugate of LPG and PAA has an antileishmanial vaccine effect against visceral leishmaniasis. In this respect, the present study may lead to new vaccine approaches based on high immunogenic LPG molecule and adjuvant polymers in fighting against Leishmania infection.

Human olfactory stem cells for injured facial nerve reconstruction in a rat model.

BACKGROUND: The purpose of this study was to show the efficacy of olfactory stem cells for injured facial nerve reconstruction in a rat model. METHODS: Olfactory stem cells were isolated from the olfactory mucosa of human participants. A 2-mm excision was performed on the right facial nerve of all rats. Reconstruction was performed with a conduit in group 1 (n = 9); a conduit and phosphate-buffered saline in group 2 (n = 9); and a conduit and labeled olfactory stem cell in group 3 (n = 9). Rats were followed for whisker movements and electroneuronography (ENoG) analyses. RESULTS: The whisker-movement scores for group 3 were significantly different from other groups (p < .001). ENoG showed that the amplitude values for group 3 were significantly different from group 1 and group 2 (p = .030; p < .001). Group 3 showed marked olfactory stem cell under a fluorescence microscope. CONCLUSION: This study suggests that olfactory stem cells may be used as a potent cellular therapy for accelerating the regeneration of peripheral nerve injuries. © 2016 Wiley Periodicals, Inc. Head Neck 38: E2011-E2020, 2016.

Isolation and diagnosis of Helicobacter pylori by a new method: microcapillary culture.

AIM: To investigate the performance of the microcapillary culture method (MCM) in Helicobacter pylori (H. pylori) isolation and diagnosis. METHODS: Microcapillary culture (MC), classical culture (CC), rapid urease (CLO) test, and histopathologic examination (HE) were performed with biopsy samples. Homogenized biopsy samples were loaded into capillary tubes and incubated for 48 h at 37 °C without providing a microaerophilic environment. Additionally, three or four loops of the homogenized sample were inoculated in a ready-to-use selective medium (Becton Dickinson, Helicobacter Agar, Modified) specific for the isolation of H. pylori and incubated at 37 °C in a microaerophilic atmosphere provided by CampyGen (Becton Dickinson, GasPack). Bacteria reproducing in microcapillary tubes were evaluated in an inverted microscope and also were evaluated after performing a CC with the content. Results obtained by CC, CLO test, and HE were compared with those of MC. The diagnostic performances of the methods used in this study were evaluated for specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and CI. RESULTS: H. pylori was found positive by CLO test + HE and/or CC culture in 26 patient antrum and corpus biopsy samples. In 25 (25/26) patient biopsy samples, H. pylori was isolated by MCM, whereas in only 14 (14/26) patient biopsy samples, H. pylori was isolated by CC. CLO test and HE were found positive in 17 (17/26) patient biopsy samples. Comparing the results of the isolation of H. pylori by MCM, CC, CLO test, and HE, the sensitivity of the MCM was found as 96%, the specificity as 80%, the PPV as 83%, the NPV as 95%, and the 95%CI as 0.76 (χ (2) = 31.51, P < 0.01) whereas the sensitivity of the CC was found as 54% (χ (2) = 19.15, P < 0.01), and the sensitivity of the CLO test and HE were found as 65% (χ (2) = 25.26, P < 0.01). CONCLUSION: This new microcapillary cultivation method for H. pylori has high diagnostic sensitivity compared with CC, HE, and CLO tests.

Utility of the microculture method in non-invasive samples obtained from an experimental murine model with asymptomatic leishmaniasis.

The sensitivity of diagnostic methods for visceral leishmaniasis (VL) decreases because of the low number of parasites and antibody amounts in asymptomatic healthy donors who are not suitable for invasive sample acquisition procedures. Therefore, new studies are urgently needed to improve the sensitivity and specificity of the diagnostic approaches in non-invasive samples. In this study, the sensitivity of the microculture method (MCM) was compared with polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescent antibody test (IFAT) methods in an experimental murine model with asymptomatic leishmaniasis. Results showed that the percent of positive samples in ELISA, IFAT, and peripheral blood (PB) -PCR tests were 17.64%, 8.82%, and 5.88%, respectively, whereas 100% positive results were obtained with MCM and MCM-PCR methods. Thus, this study, for the first time, showed that MCM is more sensitive, specific, and economic than other methods, and the sensitivity of PCR that was performed to samples obtained from MCM was higher than sensitivity of the PCR method sampled by PB.

Detection of antileishmanial antibodies in blood sampled from blood bank donors in Istanbul.

AIMS: According to the WHO, only 5-20% of the total cases of leishmaniasis are symptomatic leishmaniasis; the other cases are identified as asymptomatic leishmaniasis. In recent studies, it has been demonstrated that donor blood plays an important role in the epidemiology of asymptomatic leishmaniasis. However, the number of the studies on this subject is still insufficient. Additionally, donor blood samples obtained from Istanbul, which is the biggest metropolitan area in Turkey, have not been investigated with regard to Leishmania. Moreover, there is no information about the sensitivity of noninvasive serological methods that are used in the detection of leishmaniasis donor blood samples. Accordingly, this study aimed to investigate the presence of antileishmanial antibodies in blood samples obtained from blood bank donors in Istanbul, by using different serologic methods, and to determine the most sensitive detection method. MATERIALS & METHODS: Blood samples were taken from 188 healthy blood bank donors to the Capa Turkish Red Crescent Blood Bank (Istanbul, Turkey), and the presence of antileishmanial antibodies was measured by indirect immunofluorescent antibody test (IFAT), ELISA, immunochromatographic dipstick rapid test, and western blot (WB). RESULTS: Antileishmanial antibodies were determined in 12 out of 188 samples by IFAT (6.4%), and six out of these 12 donors were found to be positive at diagnostic titer 1:128 (3.2%). One hundred and eighty eight samples were investigated by ELISA and one (0.5%) of them gave a positive result. None of 188 samples provided a positive result by immunochromatographic test. WB applied to the 12 seroreactive donors showed that three out of 12 donors were positive. CONCLUSION: In this study, the presence of antileishmanial antibodies in blood samples of blood bank donors from Istanbul has been demonstrated by using feasible and low-cost serological methods. Additionally, in comparison with other simple and low-cost detection methods, WB was used for confirmation. IFAT has a higher sensitivity and therefore may be preferred as a prescreening method in endemic or nonendemic areas.

Leishmania tropica: the effect of darkness and light on biological activities in vitro.

Leishmania parasites can be exposed to effects of light in their vectors and hosts, at various periods. However, there is no information about the effects of light on Leishmania parasites. The aim of this study is to investigate the effects of light on various cell parameters of Leishmania tropica, in vitro. All experiments were conducted on L. tropica promastigotes and amastigote-macrophage cultures, using flow cytometric analysis, MTT and phenol-sulfuric acid assay, DAPI and Giemsa. The results showed that the morphology of parasites has changed; the cell cycle has been affected and this caused parasites to remain at G0/G1 phase. Furthermore the proliferation, infectivity, glucose consumption and mitochondrial dehydrogenase activities of parasites were decreased. Thus, for the first time, in this study, the effects of light on biological activities of Leishmania parasites were shown. These new information about parasites' biology, would be very important to investigate the effects of light on the parasites in infected vectors and hosts.

[Approaches and problems in vaccine development against leishmaniasis]. / Leishmaniasise Karsi Asi Gelistirilmesinde Yaklasimlar ve Problemler.

Leishmaniasis is a major public health problem of the world and Turkey. Recently there has been increasing interest in vaccine studies among strategies for control of leishmaniasis. Recently the increase of interest in vaccine studies among leishmaniasis control strategies makes the subject more up to date. So the aim of this review is to present information about recent vaccine studies, problems and new strategies for vaccine development studies. There are 3 generations of vaccine against leishmaniasis. First-generation vaccines are killed or live attenuated parasites; second-generation vaccines are recombinant or native antigens and live genetically modified parasites (knock out and suicidal cassettes), third generation vaccines are DNA vaccines. Also vector salivary proteins, dendritic cells and non-pathogenic L. tarentolae have been used as vaccine candidates. However there is still no effective vaccine against leishmaniasis. Since polymer conjugates considerably increase immunogenicity, polymer based vaccine studies have gained importance in recent years. However, there has not been such a study for an antileishmanial vaccine yet. LPG, surface antigen of Leishmania promastigotes, and polymer conjugates may be promising in antileishmanial vaccine studies so we are carrying out a TUBITAK Project on this subject which has been given the number, 1085170SBAG-4007.