Cell Population Doubling Time Research Articles

Impact of age and donor sites on bioactivities of tendon cells in autologous tenocyte implantation (OrthoATI™) for treatment of chronic tendinopathy

ObjectivesAutologous tenocyte implantation (OrthoATI™) therapy has demonstrated efficacy in treating patients with tendinopathy at various anatomical sites. This study evaluates the effect of patient age, gender, and tendon biopsy site on morphology, growth, and gene expression of autologous tendon cells used to treat chronic tendinopathy. MethodsPatients undergoing OrthoATI™ for tendinopathies between 2020 and 2022 were initially treated by biopsies taken from patella tendon (PT) or palmaris longus tendon (PL). The biopsies were sent to a Good Manufacturing Practice (GMP) cell laboratory where tendon cells were isolated, cultured, and expanded for four to six weeks. Cell morphology was assessed using phase contrast microscopy. Droplet digital PCR (ddPCR) was utilized for gene expression analysis. Dichotomous results were compared between groups using x2 or Fisher's exact tests with no adjustment for multiple comparisons. The nonparametric Mann–Whitney U and Kruskal–Wallis tests were utilized for the sex and age (<35y, 35-44y, 45-54y, >55y) analyses, respectively. All analyses were performed using IBM SPSS v27, and a two-tailed P-value of <0.05 was considered statistically significant. Results149 patients were included in the analysis. The PT was biopsied in 63 patients, and PL in 86 patients. There were no observer effects for age and gender between the PT and PL groups. There was no statistical significance between the PT and PL tendons for cell morphology, average cell population doubling time (PDT) (PT 83.9 vs PL 82.7 ​h, p ​= ​0.482), cellular yield (PT 16.2 vs PL 15.2 ​× ​106, p ​= ​0.099), and cell viability (PT 98.7 vs PL 99.0%, p ​= ​0.277). Additionally, ddPCR analyses showed no statistical significance found in tenogenic gene expression, including collagen type I (COL1, p ​= ​0.86), tenomodulin (TNMD, p ​= ​0.837) and scleraxis (SCX, p ​= ​0.331) between PT- and PL-derived tendon cells. An age stratification analysis found no effect on growth and gene expression. COL1 was found to be higher in males when compared to females (P ​< ​0.001), but otherwise no difference was seen in growth and gene expression in the gender analysis. No postbiopsy clinical complications were reported for either group. ConclusionThis study has shown that the growth and bioactivities of tendon cells from tendon biopsies for OrthoATI™ are not affected by tendon donor site and age. Level of evidenceIV.

Establishment of Big Bone chicken fibroblast cell bank and study of its biological characteristics

A fibroblast bank of Big Bone chicken was established using tissue adherent culture method. This cell bank included 29 embryo samples, had stocks of 147 cryogenically-preserved vials each containing 2~3Å~106 cells, and met all the cell line quality control standards established by the American Type Culture Collection (ATCC). The cells cultured in vitro showed the typical morphology of fibroblasts. The growth curve assumed the “S” shape, and the cell population doubling time (PDT) was about 30h. Cell viability was 98.1% before cryopreservation and 96.6% after recovery. All tests for microbial contamination were negative. The isoenzyme pattern was of species specificity. The frequency of diploid cells was 91%. The transfection efficiency of three fluorescent protein genes fluctuated between 10.6% and 26.5%. These results showed that the cells cultured in vitro grew well and had stable genetic properties. This research thus does not only preserve the poultry genetic resources of Big Bone chicken at the cell level, but also opens new ways for preserving important genetic resources of endangered animals in the form of somatic cells.

Long-term preservation of established fibroblast lines from six-banded armadillos (Euphractus sexcintus, Linnaeus, 1758) by extended passage and cryopreservation.

Establishing new somatic cell cultures has raised significant attention as an effective and convenient way to preserve genetic samples for different applications. Although many lines have been established in model animals, none derived from six-banded armadillo species is currently available. We report the successful isolation and characterization of fibroblasts from six-banded armadillos, evaluating the cell quality after extended culture and cryopreservation. Initially, we collected ear skin from five captive adult individuals and identified fibroblast lines by morphology, karyotyping, and immunophenotyping assays. The isolated fibroblasts were evaluated after several passages (fourth, seventh, and tenth passages) and cryopreservation by slow freezing. Cell morphology, viability, metabolism, proliferative activity, mitochondrial membrane potential, and apoptosis levels were analyzed. The skin explants had great adhesion, and cell outgrowth could be seen after 3-6 d. The cells were verified as fibroblasts at the fourth passage by vimentin expression and normal karyotype (2n = 58). The viability remained high (> 87%) and constant from the fourth to the tenth passage (p > 0.05). The passages did not change the cell morphology and metabolic and growth rates. Moreover, cryopreservation did not affect most evaluated parameters; post-thawed cells maintained their viability, growth, metabolism, and apoptosis levels. Nevertheless, cryopreservation increased mitochondrial membrane permeability and cell population doubling time compared to non-cryopreserved cells (p < 0.05). In summary, viable fibroblasts can be obtained from six-banded armadillo skin while conserving their quality as the number of passages increases and featuring few changes after cryopreservation.

Fabrication of elliptically constructed liquid crystalline elastomeric scaffolds for 3D artificial tissues

Inspired by the orientation and the fibrous structure of human muscle tissues, we fabricated preconstructed porous liquid crystalline (LC) scaffolds through a two-step polymerization and salt leaching method. A novel strategy combining the aligning properties of LCs and the ease of processing of elastomers for the preparation of elliptical scaffolds for muscle cell culture was proposed in this research. Different from the other types of scaffolds, our biocompatible LC scaffold that can be implanted into the human body using a supporting unit to improve the mechanical properties compared with those of natural muscle. To evaluate the synthesized scaffolds, in vitro experiments using normal human dermal fibroblast (NHDF) cells and smooth muscle cells from rats were carried out, and the sample cells were cultured on each sample scaffold. Based on the results of long-term culture of NHDF cells on the LC scaffolds, it can be confirmed that all three kinds of LC scaffolds have good biocompatibility and provide enough space for cell growth. The addition of gelatin can significantly enhance the biocompatibility of the synthesized scaffolds. Evaluation of scaffold morphologies on cell growth indicates that the molecular arrangement on the scaffolds can induce the growth direction of smooth muscle cells to a certain extent, thereby increasing the formation of highly ordered arrangement tissues. The population doubling time of NHDF cells on the different scaffolds suggest that gelatin can improve the attachment and growth of cells. Investigation of cell viability on LC scaffolds shows that the original LC scaffolds already possess excellent biocompatibility. Additionally, the average cell viability of smooth muscle cells was above 90%, showing that the LC scaffolds in this research are suitable for application in muscle tissue engineering. Based on the results, the gelatin-coated scaffolds are more conducive to the growth of cells in this research and provide promising candidates for tissue engineering in biomedical fields and research fields.

Genotoxicity assessment of root extracts of Paeonia lactiflora Pall

The roots of Paeonia lactiflora Pall., (Paeoniae Radix, PL) are a well-known herbal remedy used to treat fever, rheumatoid arthritis, systemic lupus erythematosus, hepatitis, and gynecological disorders in East Asia. Here we evaluated the genetic toxicity of PL extracts (as a powder [PL-P] and hot-water extract [PL-W]) in accordance with the Organization for Economic Co-operation and Development guidelines. The Ames test revealed that PL-W was not toxic to S. typhimurium strains and E. coli in absence and presence of the S9 metabolic activation system at concentrations up to 5000μg/plate, but PL-P produced a mutagenic response to TA100 in the absence of S9 mix. PL-P was cytotoxic in in vitro chromosomal aberrations (more than a 50% decrease in cell population doubling time), and it increased the frequency of structural and numerical aberrations in absence and presence of S9 mix in a concentration-dependent manner. PL-W was cytotoxic in the in vitro chromosomal aberration tests (more than a 50% decrease in cell population doubling time) only in the absence of S9 mix, and it induced structural aberrations only in the presence of S9 mix. PL-P and PL-W did not produce toxic response during the in vivo micronucleus test after oral administration to ICR mice and did not induce positive results in the in vivo Pig-a gene mutation and comet assays after oral administration to SD rats. Although PL-P showed genotoxic in two in vitro tests, the results from physiologically relevant in vivo Pig-a gene mutation and comet assays illustrated that PL-P and PL-W does not cause genotoxic effects in rodents.

Determining the Population Doubling Time of HepG2 and Huh-7 Cells and the Toxic Effect of Dimethyl Sulfoxide (DMSO)

Developing cell lines that carry promising qualities closest to human hepatocytes in drug studies is a dream of many research laboratories. About 90% of drugs are metabolised by the liver. Therefore, most drug discovery studies utilise hepatocytes to understand the basic mechanism of the drug’s breakdown. To assure hepatocytes survival, various solvents were tested for their cytotoxicity on the cell line. Since most drugs are weakly soluble in water, they can be dissolved in an aprotic solvent. To obtain accurate results, the requirements of these solvents are biocompatible and non-toxic to the cells. One of the most commonly used solvents is dimethyl sulfoxide (DMSO). This study aims to understand the cell characteristic of cells doubling time and investigate the toxic effect of DMSO using the cell proliferation measurement technique. The toxicity was measured on liver-derived cell lines HepG2 and Huh-7. Cell growth and morphology were observed with an inverted phase microscope while cell viability was counted using Vi-Call XR 2.03. Results showed that a doubling time of 32 hours would be best for treatments on both cells. A concentration of more than 0.40% DMSO has significant toxicity and caused inhibition of proliferation on both cell lines, resulting in low cell count and higher cell death. Ethanol and methanol were observed as good solvents since they have low toxicity. However, drugs that were only dissolved in a low concentration of DMSO (0.05-0.2%) gave the best result without any harmful effect on cell proliferation.&#x0D;

In vitro Culture and Morphometry of Porcine Adipose Derived Mesenchymal Stem Cells (pAD-MSCs)

Background: Mesenchymal stem cells are well known for their self-renewal capacity and ability to differentiate into multiple cell lineages. The aim of the study was to develop a simple technique for isolation of mesenchymal stem cells from porcine adipose tissue and to study the morphometric characteristics of porcine mesenchymal stem cells. Methods: Porcine adipose derived mesenchymal stem cells were isolated in vitro by using collagenase type II enzyme. Cell yield and viability of the cells were calculated by using trypan blue exclusion method using Neubauer’s chamber. Characterization of MSCs were done by using specific cell markers. The morphological changes, morphometry were analysed in culture using Leishman’s stain. The cell doubling (CD) and Population doubling time (PDT) were also calculated. Result: The isolated adherent cells start forming colony and demonstrated an elongated, round and spindle like fibroblastic morphology by day 1. Almost 80-90 per cent confluency was attained on day 8-9 after the initial seeding and was reduced to day 3-4 in the subsequent passages. RT-PCR reactions revealed positive expression of mesenchymal stem cell markers CD44, CD73 and negative expression of CD34, a hematopoietic cell surface marker. Immunocytochemistry also revealed positive expression for CD44 and negative for CD34. In morphometric studies, the cell length, nucleus length, cell width and nucleus width were increased between 24 and 48 hours in both P2 and P3.

Characterization of mesenchymal stem cells isolated from Wharton\u2019s jelly of the human umbilical cord

BackgroundIsolation of post-partum umbilical cord Wharton’s jelly stem cells has gained attention as an alternative source of the bone marrow. Because easy isolation, lack of ethical concerns, and the presence of both embryonic and adult stem cells have made them a valuable source for use in therapeutic applications and regenerative medicine. The study utilized a modified protocol using in-house human pooled cord blood serum for isolation and expansion of the mesenchymal stem cells obtained from the human umbilical cord Wharton’s jelly. Cell proliferation and population doubling time and tri-lineage differentiation were assessed, and the expressions of mesenchymal cell surface markers CD44, CD90, CD105, and CD34 were assessed by flow cytometry and RT-PCR. The genetic stability of the isolated cells was assessed by chromosomal karyotype.ResultsThe isolated cells displayed fibroblastic-like morphology and tri-lineage differentiation into adipocyte, chondrocyte, and osteocyte. The isolated cells maintained the proliferative competence with a doubling time ranged from 38 to 42h and corresponded well with the standard positive and negative molecular markers (CD44+, CD90+, CD 105+, and CD34−). Cell senescence occurred at the later passage of the cells (P15) affecting, about 25% of the population. Metaphases spread of the cells showed normal diploid karyotypes, with typical chromosomal plates indicating genetic stability of the isolated cells.ConclusionThe primary cultures exhibited success in isolating the umbilical cord Wharton’s jelly mesenchymal stem cells, which maintained their tri-lineage differentiation potential, phenotypes and karyotype characteristics on further passage and expansion.

Differential Development of Umbilical Cord-Derived Mesenchymal Stem Cells During Long-Term Maintenance in Fetal Bovine Serum-Supplemented Medium and Xeno- and Serum-Free Culture Medium

Umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs) are believed to have potential for the treatment of various diseases; thus, many scientists have investigated the molecular mechanisms underlying the function of UC-MSCs and, for example, the appropriate media for large-scale UC-MSC expansion to prepare cells for real-world application. In this study, we investigated the cellular morphology, proliferation capacity, surface markers, cellular senescence signals, clonogenic potential, trilineage differentiation capacity, and secreted factors of human primary UC-MSCs in long-term culture from passage 2 (P2) to passage 10 (P10) with either conventional fetal bovine serum (FBS)-supplemented medium or commercial xeno- and serum-free medium (StemMACS™). We found that the cells cultured in both media had similar morphology and marker expression. However, the proliferation kinetics as measured by the cell population doubling time differed in a passage (P2-P10)-dependent manner between the cells cultured in the two media; sustainable growth was observed in cells maintained in xeno- and serum-free medium. Moreover, significant differences in cellular senescence signals were observed, with more aging cells in the cell population cultured in FBS-containing medium. Colony numbers and the day that the first colony appeared were similar; however, UC-MSC colony sizes were smaller when cultured in FBS-containing medium. In addition, the multidifferentiation potential of UC-MSCs cultured in xeno- and serum-free StemMACS medium was maintained during long-term culture, but this potential was lost for adipogenic differentiation at P9. Moreover, secreted epidermal growth factor and vascular endothelial growth factor (VEGF)-A were detected in the conditioned media from UC-MSCs, whereas platelet-derived growth factor was not. Similar expression of these factors was observed in conditioned media of UC-MSCs cultured in StemMACS, but the VEGF level was higher in young UC-MSCs (P6) than in aged UC-MSCs cultured in FBS-supplemented Dulbecco's modified Eagle's medium/F12. Thus, StemMACS is better for UC-MSC expansion than conventional FBS-supplemented culture medium, especially when culturing UC-MSCs for real-world applications.

Multifarious Functions of Butyrylcholinesterase in Neuroblastoma: Impact of BCHE Deletion on the Neuroblastoma Growth In Vitro and In Vivo.

The physiological functions of butyrylcholinesterase (BChE) and its role in malignancy remain unexplained. Our studies in children newly diagnosed with neuroblastoma indicated that BChE expressions is proportional to MYCN amplification suggesting that pathogenesis of high-risk disease may be related to the persistent expression of abnormally high levels of tumor-associated BChE. BChE-deficient neuroblastoma cells (KO [knockout]) were produced from MYCN -amplified BE(2)-C cells (WT [wild-type]) by the CRISPR-Cas9 targeted disruption of the BCHE locus. KO cells have no detectable BChE activity. The compensatory acetylcholinesterase activity was not detected. The average population doubling time of KO cells is 47.0±2.4 hours, >2× longer than WT cells. Reduced proliferation rates of KO cells were accompanied by the loss of N-Myc protein and a significant deactivation of tyrosine kinase receptors associated with the aggressive neuroblastoma phenotype including Ros1, TrkB, and Ltk. Tumorigenicity of WT and KO cells in male mice was essentially identical. In contrast, KO xenografts in female mice were very small (0.37±0.10 g), ~3× smaller compared with WT xenografts (1.11±0.30 g). Unexpectedly, KO xenografts produced changes in plasma BChE similarly to WT tumors but lesser in magnitude. The disruption of BCHE locus in MYCN -amplified neuroblastoma cells decelerates proliferation and produces neuroblastoma cells that are less aggressive in female mice.

Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes.

Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

ESTABLISHMENT AND CRYOPRESERVATION OF FIBROBLAST CELL LINE FROM A SUMATRAN RHINOCEROS (Dicerorhinus sumatrensis)

Cell lines have been established to preserve the genetic material of endangered animals. This study aims to establish, characterize and authenticate fibroblast cells derived from the kidney tissue of a Sumatran rhinoceros’ carcass. The primary cultures were obtained using the mixed enzymatic-explant method, supplemented with complete media and maintained at 37oC with 5% CO2 in an incubator. Following routine trypsinization, viability and growth curves were generated through the Trypan Blue counting method. Cellular senescence was quantified by Sa-β-gal staining assay and G-banding for karyotyping. As a result, the cell derivation had generated 81 frozen stocks. The viability of cells at P5 and P10 showed reasonable recovery after six months. Cell population doubling time at P5 was 20.45 hours, while it was 22.35 hours at P10 and P15. The senescence level significantly increased from P5 to P10, and was especially significant at P15. Genetic stabilities were considered stable at P5 and P10, with frequency of over 70 %. In conclusion, this study was able to derive a primary fibroblast culture from the preserved tissue of a Sumatran rhinoceros, with certain changes in morphology, senescence level, growth curves and cell viability as the number of passages increased.

Pharmacological effects of active saponins from Panax ginseng Meyer

Purpose: To investigate the pharmacological effects of the active saponins isolated from Panax ginseng Meyer (P. ginseng) via extraction, heat treatment, and enzyme conversion.&#x0D; Methods: The effects of active saponins on rat blood were determined using a multichannel analyzer. The population doubling time (PDT) of mesenchymal stem cells (MSCs) and human-derived leukocyte cancer cells (A549) was determined by cell counting. b-galactosidase was measured in human toothderived stem cells (HTS) using a β-galactosidase ELISA kit.&#x0D; Results: Intraperitoneal administration of active saponins resulted in 30.09 % increase in red blood cell count and 55.55 % decrease in blood triglyceride concentrations. The stimulatory effect of active saponins (10 ng/mL) on cellular differentiation was determined based on PDT of MSCs, which decreased by 33.82 % compared to control. A 22.29 % increase in PDT of A549 cells demonstrated the suppressive effects of active saponins on cancer cell growth. Active saponins (10 ng/mL) also decreased intracellular β-galactosidase concentration by 20.42 % in HTS cells.&#x0D; Conclusion: Administration of active saponins to rats extends the lifespan, promotes differentiation in MSCs, suppresses A549 cell differentiation, and reduces TG and b-galactosidase associated with aging in HTS. Thus, active saponins have potentially beneficial effects in humans.

Effects of human platelet lysate on the growth of cultured human corneal endothelial cells

Human platelet lysate (hPL) as a replacement for foetal bovine serum (FBS) in culturing human corneal endothelium is an emerging area of interest, although there are limited studies evaluating the quality of the hPL being used. Our study aimed to evaluate variations between sources of hPL and to explore the efficacy of hPL (with and without heparin) as a replacement for FBS in culturing human corneal endothelial cells in vitro. Immortalized human corneal endothelial cells (B4G12) and primary human corneal endothelial cells (PHCEnCs, n = 11 donors, age from 36 to 85 years old) were cultured with 5% hPL or FBS. A full characterisation of the effects of hPL and FBS on cell growth was conducted using IncuCyte Zoom (percentage cell confluence and population doubling time, PDT) to analyse cell proliferation. AlamarBlue assays were used to measure cell viability. The concentration of fibrinogen, PDGF, hEGF, VEGF and bFGF in two sources of hPL were analyzed by Enzyme-linked immunosorbent assay. Expression and localization of Na+/K+-ATPase, ZO-1 and CD166 on PHCEnCs and B4G12 cells were assessed with immunofluorescence and immunoblotting. Our results showed that a significant difference in fibrinogen, hEGF and VEGF concentrations was found between two sources of hPL. Heparin impaired the positive effect of hPL on cell growth. PDT and alamarBlue showed that hPL significantly increased proliferation and viability of PHCEnCs in two of three donors, and immunostaining indicated that hPL increased ZO-1 and CD166 expression but not Na+/K+-ATPase on PHCEnCs. In addition, heterogeneities on immunopositivity of Na+/K+-ATPase and ZO-1 and morphology were found on PHCEnCs derived from an individual donor cultured with hPL medium. In conclusion, hPL showed positive effect on primary corneal endothelial cell growth, and maintenance of their cellular characteristics compared to FBS. hPL can be considered as a supplement to replace FBS in PHCEnC culture. However, the variation observed between different hPL sources suggests that a standard quality control monitoring system such as storage time and a minimal concentration of growth factors may need to be established.

Investigation the Effect of Serum Rich in Growth Factors on Proliferation, Growth and Expression of Genes Involved in Cell Longevity by Mesenchymal Stem Cells

Background: The limited lifespan of Mesenchymal Stem Cells (MSCs) has highly restricted their application. The Serum Rich in Growth Factors (SRGF) contains growth factors that are involved in processes of the growth and proliferation of MSCs. The present study was aimed to examine the regulatory effects of SRGF on the expression of some genes which effect both proliferation and lifespan of MSCs. Methods: SRGF was obtained from platelets and MSCs were isolated from umbilical cord. The MSCs morphology and phenotype have been analyzed using phase-contrast microscope and flow cytometry, respectively. Cells were cultured either in presence of FBS 10% (as control) or SRGF 5% plus FBS 5% and FBS 10% alone (as tests). The cell Population Doubling Time (PDT) was measured hourly. The expression of related genes was analyzed employing real-time PCR technique. Findings: Finding of the present study showed that the experimental groups were morphologically and phenotypically as similar as to control group. We observed that the PDT in the experimental group was shorter than that was found in the control. We have also found that the expression of hTERT and c-MYC genes was increased, while, and P16 and P53 genes were down- regulated. These results were superior in the 10%SRGF group than in the 5% SRGF + 5% FBS group. Conclusion: According to the finding of this study, SRGF could possibly serve as an effective proliferative and lifespan inducing factor for MSCs. These results also indicated that SRGF has the tendency to be employed as an appropriated alternative for FBS in cell culture.

DCLK1, a Putative Stem Cell Marker in Human Cholangiocarcinoma.

Background and AimsCholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin‐like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA).Approach and ResultsSpecimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 [leucine‐rich repeat‐containing G protein‐coupled receptor], CD [clusters of differentiation] 90, EpCAM [epithelial cell adhesion molecule], CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real‐time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2‐IN‐1) on cell proliferation (MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium] assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real‐time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme‐linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared with unsorted cells. LRRK2‐IN‐1 showed an anti‐proliferative effect in a dose‐dependent manner. LRRK2‐IN‐1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls.ConclusionsDCLK1 characterizes a specific CSC subpopulation of iCCACD133+ and pCCALGR5+, and its inhibition exerts anti‐neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.

In vitro Cytotoxicity Analysis of Hybrid Graphene Oxide (hGO) Nano Structures in Caprine Wharton’s Jelly Derived Mesenchymal Stem Cells (WJ-MSCs)

Background: Nanotechnology is used in stem cell culture as well as in vivo delivery and tracking of stem cells. Graphene oxide (GO) is a carbon based nanomaterial and it has large surface area as well as good biocompatibility and heteroatoms doped GO exploit its properties. Hybrid GO (hGO) nano structures biocompatibility is depends on its size, dose and exposure time as well as in vitro cell models and hence, need thorough cytotoxicity studies in different species in vitro cell models. Methods: Caprine Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) were isolated, characterized and dose dependent (100, 50, 25, 10 and 0µg /ml) in vitro cytotoxicity of three different hGO nano structures (phosphorus doped graphene oxide titanium oxide tubes, rods and sheets) were analysed in caprine WJ-MSCs by studying cell cytotoxicity assays. Result: All three hGO nano structures were damaged cell morphology at 100 and 50 µg /ml doses, however, morphologically more good cells were observed in hGO tubes treated group than hGO rods and hGO sheets at 25 and 10 µg/ml doses as compared to control. Cell viability percentage was significantly (P less than 0.01) decreased at dose 100 µg/ml and it was significantly (P less than 0.01) increased at 25 µg/ml dose as compared to 50, 10 and 0 µg/ml doses. But, hGO tubes significantly (P less than 0.01) increased cell viability % as compared to hGO rods and hGO sheets. Cell population doubling time (PDT) was not altered significantly by all hGO nano structures, but 100 and 50 µg/ml doses significantly (P less than 0.01)increased cell PDT as compared to 25, 10 and 0 µg/ml doses. All hGO nano structures were non significantly altered growth curve, however, all hGO nano structures at 25 µg /ml dose altered (inclined) shape of growth curve, while 100 and 50 µg /ml doses significantly declined growth curve shape as compared to 10 and 0 µg /ml doses. Cell proliferation % was significantly (P less than 0.01) increased at 25 and 10 µg/ml doses, while, it was significantly (P less than 0.01) decreased at 100 µg /ml dose as compared to 50 and 0 µg /ml. However, there was no significance difference was observed in cell proliferation % in groups treated by different hGO nanostructures. In last, it was concluded as, hGO nano structures cytotoxicity was dose dependent and hGO nano tubes were least cytotoxic in caprine WJ-MSCs.

Establishment and characterization of a new embryonic cell line from Helicoverpa armigera (Hübner).

Here, a new cell line, Ha168, was established from Helicoverpa armigera eggs and has been stably subcultured for over 30 passages in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line consists of round and spindle-shaped cells and several giant cells. The round cells, with a cell diameter of 14.30 ± 2.804μm, account for 77% of the cells. DNA amplification fingerprinting, random amplified polymorphic DNA analysis, and analysis of the mitochondrial cytochrome c oxidase subunit I gene confirmed that the Ha168 cells were derived from H. armigera. Karyotype analysis revealed the average chromosome number of Ha168 cells to be 71. Growth curves at passage 25 were determined and demonstrated that the cell population doubling time is 56.8h. No mycoplasma contamination was detected in the cell line. Ha168 cells can be infected by recombinant baculovirus AcMNPV-EGFP, and exogenous protein expression level in this cell line is 70% of that in the Sf9 cell line.

Human Platelet Releasate (hPR) - A xeno-free supplement for the expansion of stem cells

Background & Aim Background Recent regulatory guidelines for cell therapy applications recommend the use of xeno-free materials to mitigate the risk of immune reaction to animal-derived additives i.e. fetal bovine serum (FBS). On this note, human platelet lysate (hPL) has been accepted as a xeno-free alternative for stem cell expansion. With several methods available to manufacture hPL, the lack of standardization amongst manufacturers has led to inconsistencies in expansion rate, phenotype, and immunomodulatory properties of stem cells; altering the intrinsic therapeutic potential of cells. Further, some platelet lysates induce “conditioning of cells”, where the cell expansion occurs with a bias towards a lineage of differentiation, as directed by the composition and concentration of growth factors, cytokines and chemokines present in the hPL. We have developed a cGMP process to manufacture human platelet releasate (hPR), a potential alternative to hPL and FBS. hPR is a xeno-free, non-conditioning media supplement derived from expired-human platelets. Methods, Results & Conclusion Methods We have developed a proprietary closed-system manufacturing process designed for optimal extraction of growth factors, cytokines and chemokines (Fig.1). Every lot is tested for sterility, endotoxin, mycoplasma, total protein content, and growth factors. The performance characteristics was evaluated by expanding bone marrow derived MSCs (BM-MSCs) in basal growth medium (DMEM) supplemented with hPR and benchmarked with equivalent strengths of three commercially available non-heparin-requiring human platelet lysates, AB serum, and FBS. Cell proliferation and population doubling time were measured using Luna II automated cell counter. Tri-lineage differentiation potential of these cells was assessed with adipogenesis, chondrogenesis and osteogenesis assay. The phenotypic, and biochemical markers of BM-MSCs were quantified using flow cytometry and multiplex ELISA quantibody assay (Fig.2). The potency of expanded cells was assessed by i) measuring human indoleamine 2, 3-dioxygenase (IDO) using kynurenine assay and luminex assay (IDO human ProcartaPlex), ii) mixed lymphocyte reaction assay (a lab-developed co-culture assay), and regulatory T-cell conversion assay using flow cytometry. Results We have developed a cGMP-compliant process to manufacture consistent lots of hPR. Our results indicate that the potency, and proliferation rate of MSCs is higher in basal medium supplemented with hPR compared to FBS or hPL (Fig.3).

Abstract 2554: Branched chain amino acid transaminase 1 in claudin-low breast cancer

Abstract Breast cancer, the most commonly diagnosed cancer in women, can be classified into five distinct subtypes. One subtype, claudin-low breast cancer, accounts for approximately 7% of the breast cancer cases and these tumors are aggressive and highly metastatic. RNA sequencing of human claudin-low breast cancers by other groups and RNA sequencing of a murine claudin-low mammary tumor cell line by our group has revealed that Bcat1 is significantly up-regulated in this breast cancer subtype. Bcat1 regulates the metabolism of branched chain amino acids and has been linked to numerous pathologies including heart disease, diabetes, and cancer. Based on this data we hypothesized that the expression of Bcat1 in claudin-low mammary tumors is driving the aggressive and metastatic nature of this cancer subtype and disrupting Bcat1 will deter these features. Elevated expression of Bcat1 in the murine claudin-low cell line RJ423, compared to the murine luminal mammary tumor cell line RJ345, has been confirmed at the mRNA and protein level. Bcat1 has been transiently down-regulated ~70% in RJ423 cells using siRNA and this suppression of Bcat1, contrary to the anticipated result, showed no effect on proliferation based on phospho-histone H3 immunofluorescence. However, when the cell population doubling time was investigated, a significant difference was seen between the RJ423 cell line and the cell lines with suppressed Bcat1 expression. To elucidate these contradicting results, cell cycle analysis using Bromodeoxyuridine and 7-AAD through flow cytometry is currently underway. RJ423 stably expressing Bcat1 shRNA and RJ345 stably expressing a Bcat1 expression vector are currently being selected. Further in vitro and in vivo studies will utilize these stably transfected cell lines. This study will determine whether further investigation into the effects of Bcat1 on claudin-low human breast cancer is prudent and if Bcat1 may be used as a therapeutic target. Citation Format: Lisa C. Reynen, Robert Jones, Roger Moorehead. Branched chain amino acid transaminase 1 in claudin-low breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2554.