Time-lapse Imaging System Research Articles

Advantages of a Time-Lapse Imaging System in Assisted Reproductive Technology

Abstract: Clinical use of a time-lapse system and the develpoment of continuous culture media have enabled the establishment of modern incubation systems which decrease stress on the embryo. Furthermore, the use of a time-lapse system allows the accumulation of successive images of embryonic development. Various embryo morphological kinetics are beginning to be analyzed by using the collected data. A time-lapse system is useful for the accurate evaluation of implantation ability and deciding which embryos should be transferred first. Also, a time-lapse system allows the elimination of embryos which shoud not be transferred because of abnormal cleavage, or a multinucleated blastomere after the 4-cell stage.

Collapse of blastocysts is strongly related to lower implantation success: a time-lapse study.

Is there an association between blastocyst collapse patterns and implantation potential? Embryos that exhibit collapse are as likely to hatch as those that do not, but are less likely to implant and should not be replaced if alternatives are available. Studies of blastocyst collapse in different species of mammals have found that most blastocysts, that experience consecutive weak contractions, hatch successfully whereas those that exhibit strong contractions or collapse, fail to hatch. Retrospective cohort study. Seven hundred and fifteen transferred blastocysts were analyzed from July 2012 to May 2013. University-affiliated infertility center. Participant patients were recipients of oocyte donation and autologous IVF cycles (n = 460). Embryo development was analyzed with a time-lapse imaging system. Variables studied included blastocyst collapse (defined as the separation of ≥50% of the surface of the trophectoderm of the blastocyst from the zona pellucida), kinetic variables, embryo morphology, implantation and clinical pregnancy rates. A total of 139 blastocysts presented collapse (19.4%), of these 8 presented 2 collapses and 2 presented 3 collapses. The timings of embryo cleavages and the time taken to reach the morula stage and blastulation were significantly shorter in embryos with collapse than in those without. Implantation rates were compared in cycles where either all or none of the embryos replaced, implanted so that implantation data were known for every embryo. Among 408 embryos without collapse and 94 embryos with at least one collapse (94) the implantation rates were 48.5% (95% confidence intervals (CI) 46.7-53.4%) and 35% (95% CI 25.3-44.9%), respectively. The percentage of embryos that hatched was similar in both groups 28.7 and 31%, respectively. The retrospective nature of the study limits its potential value. Study the collapse pattern of the blastocyst, may assist selection of the blastocysts most likely to implant and increase IVF/ICSI success rates.

Euploidy is not predictable by time lapse imaging system with either blastomere or trophectoderm biopsies

Euploidy is not predictable by time lapse imaging system with either blastomere or trophectoderm biopsies

Abstract 202: Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines

Abstract Introduction: Quantitative analytical systems have the ability to quantify the amount of constituents on a per entity basis. For example, flow cytometers use stoichiometric DNA stains to quantify the amount of DNA on a per cell basis, and allow discrimination of the cell cycle phases G0/G1, S-Phase, and G2/M. Quantitative imaging cytometers employ morphometric features (total DNA content, DNA maximum brightness, and nuclear area) to track the cell cycle during the mitotic phases. As cells enter mitosis, the condensation of the chromatin to form chromosomes increases the DNA density, while the nuclear area and volume decrease. Unfortunately, DNA dyes are harmful for live cells and can interfere with the results of toxicological assays. It is desirable to have a method for label-free long-term evaluation of the cell cycle in living cells. Methods: The Holomonitor®M4 (Phase Holographic Imaging, Lund, Sweden) is an incubator-adapted time-lapse imaging system that records interference patterns from a low power 635nm diode laser. Computer software is used to de-convolve the interference pattern at a plurality of heights to form 3-dimensional and 2-dimensional images of the sample thickness. These images are segmented and features (cellular area, optical thickness and volume, etc.) are calculated on a per cell basis. Positional information is recorded for each cell. Data can be presented as positional track charts, position vs. feature graphs, and feature vs. feature graphs. We obtained time-lapse videos using proprietary PHI software and also generated 4-dimensional plots (X-position, Y- position, optical thickness coded as the pixel intensity, and time) in ImageJ software. Results: In our studies of SKOV3 tumor cell lines, we found a set of cellular based features (cell volume, optical thickness, and cell area) that correlate with the DNA-based features we are accustomed to using in our traditional label-based systems. In the 4D plots, the history of each cell within the imaging area is presented for the entire evaluation time - typically 18-24 hours with 5 minute sampling increments. With a low optical thickness threshold, tracks of all of the cells are displayed. The image intensity threshold can be raised, so only mitotic events are displayed. Mitotic cells have a characteristic Y shaped tracks, appearing suddenly as the chromatin condenses, bifurcating as the daughter cells move away from each other, and then disappearing as the chromatin de-condenses. Cells blocked in mitosis present as persistent long tracks. The rapid degradation of apoptotic cells is often visible, while cells undergoing mitotic catastrophe suddenly disappear. Conclusions: Time-lapse label-free holographic imaging combining traditional analysis and novel display techniques is well suited for toxicological studies and visualization of drug effects on the cell cycle. Citation Format: Ed Luther, Jeffrey Agar, Mansoor Amiji. Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 202. doi:10.1158/1538-7445.AM2015-202

A critical appraisal of time-lapse imaging for embryo selection: where are we and where do we need to go?

The aim of this study was to undertake a critical appraisal of the available evidence for the use of time-lapse imaging for embryo selection in clinical IVF. A literature search in PubMed, Scopus, Cochrane Central, ClinicalTrials.gov, Current Controlled Trials, and WHO International Clinical Trials Registry Platform was performed to identify randomized controlled trials that investigated the effect of time-lapse embryo selection and/or the time-lapse incubation system on ongoing pregnancy rate. We then performed a systematic review and assessed the relative risks (RRs) and 95 % confidence intervals (CIs) for ongoing pregnancy rates and the risk of bias of the eligible studies. We identified four eligible randomized studies, three of which investigated the effect of both time-lapse incubation system and selection on ongoing pregnancy rate; the pooled result revealed a benefit of this intervention (relative risk (RR) 1.20; 95 % CI 1.05-1.37). However, the evidence was judged to be of low quality due to study limitations; a beneficial effect was observed in only one study deemed to be at high risk of bias. The single study assessing the effect of only the time-lapse incubation system revealed a non-significant negative effect (RR 0.71; 95 % CI 0.49-1.03). The findings from this systematic review of the current evidence do not support routine use of time-lapse technology in clinical IVF. We therefore believe that the use of time-lapse imaging for embryo selection should remain experimental and that couples should not be subject to a surcharge for having their embryos cultured in a time-lapse imaging system. Future studies evaluating this technology in well-designed trials should be performed.

Development of a Monitoring Method for Nonlabeled Human Pluripotent Stem Cell Growth by Time-Lapse Image Analysis.

: Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy. This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs.

Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage.

What is the relationship between cleavage stage embryo kinetics, blastocyst metabolism and subsequent embryo viability? Embryos cleaving faster at the first cleavage division resulted in blastocysts with a larger inner cell mass (ICM), higher glucose consumption, lower glycolytic rate, higher aspartate uptake, lower global amino acid turnover and higher percentage of developing fetuses on E13.5 when compared with blastocysts that developed from slower cleaving embryos. Previous research has shown that morphokinetics, blastocyst carbohydrate metabolism and cleavage stage amino acid metabolism of the preimplantation embryo can be used independently as markers of its developmental competence and subsequent viability. Morphokinetics of in vitro fertilized mouse zygotes were observed using a time-lapse imaging system and they were identified as 'fast' or 'slow' cleaving embryos. Spent culture media from resultant blastocysts were analysed for carbohydrate and amino acid utilization. Blastocysts either had their ICM and trophectoderm (TE) cell number determined, were cultured further in an outgrowth assay or transferred to a recipient female to assess implantation and fetal development. Morphokinetics of in vitro fertilized C57BL/6xCBA (F1) zygotes individually cultured in 2 µl drops of G1/G2 media with HSA under Ovoil in 5% O2, 6% CO2 and 89% N2 were analysed using a time-lapse incubator. At 72 h post-insemination, blastocysts were separated into quartiles derived from timing of the first cleavage division. Blastocysts were cultured for a further 24 h and spent media samples, including controls containing no embryos, were frozen and subsequently analysed for amino acid utilization using liquid chromatography-mass spectrometry. These blastocysts were then analysed over a further 1.5 h period for carbohydrate utilization and subsequently stained to determine ICM and TE cells. To analyse implantation potential, fetal quality and viability, additional 'fast' and 'slow' blastocysts were cultured further in an outgrowth model or transferred to recipient females. Embryos cleaving faster at the time of first cleavage (first quartile, designated 'fast') were on average 2.5 h ahead of slower embryos (fourth quartile, designated 'slow', 15.1 ± 0.1 versus 17.6 ± 0.1 h, P < 0.001). On Day 5 of culture, blastocysts developed from 'fast' embryos had a larger ICM number (17.4 ± 2.1 versus 7.4 ± 2.0, P < 0.01), a higher glucose consumption (21.2 ± 1.2 versus 14.3 ± 1.0 pmol/embryo/h, P < 0.001) and a lower glycolytic rate (expressed as the percentage of glucose converted to lactate) (49.6 ± 2.8 versus 59.7 ± 2.8%, P < 0.05) compared with 'slow' embryos. Further non-invasive metabolomic analysis revealed that 'fast' blastocysts consumed more aspartate (2.2 ± 0.1 versus 1.8 ± 0.1 pmol/embryo/h, P < 0.05) and produced little or no glutamate compared with 'slow' blastocysts (0.02 ± 0.07 consumed versus 0.32 ± 0.11 pmol/embryo/h produced, P < 0.05). Transfer of 'fast' blastocysts to pseudo-pregnant recipients resulted in higher fetal survival post-implantation compared with 'slow' blastocysts (69.6 versus 40.4%, P < 0.01). The timing of the first cleavage division was used to classify blastocysts as 'fast' or 'slow' embryos; however, a combination of several developmental kinetic markers (cleavage time of 3- to 8-cell, duration between cleavage division times) may be used to more accurately determine an embryo as 'fast' or 'slow'. Only the fastest and slowest quartiles (those embryos with the fastest and slowest first cleavage division) were analysed in this study. These findings show that kinetically different embryos develop into blastocysts with different metabolic profiles and viability. Work is now being undertaken to determine if using these viability markers in combination will increase embryo selection efficacy and further improve implantation and pregnancy rates. The study was funded by the University of Melbourne. The authors have no conflicts of interest to declare.

The use of morphokinetics as a predictor of implantation: a multicentric study to define and validate an algorithm for embryo selection.

Can we use morphokinetic markers to select the embryos most likely to implant and are the results likely to be consistent across different clinics? Yes, morphokinetic markers can be used to select the embryos most likely to implant and the results were similar in different IVF clinics that share methods and organization to some extent. With the introduction of time-lapse technology several authors have proposed the use of kinetic markers to improve embryo selection. The majority of these markers can be detected as early as Day 2 of development. Morphology remains the gold standard but kinetic markers have been proven as excellent tools to complement our decisions. Nevertheless, the majority of time-lapse studies are based on small data sets deriving from one single clinic. Retrospective multicentric study of 1664 cycles of which 799 were used to develop an algorithm (Phase 1 of the study) and 865 to test its predictive power (Phase 2 of the study). University-affiliated infertility centres patients undergoing first or second ICSI cycle using their own or donated oocytes. Embryo development was analysed with a time-lapse imaging system. Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation (IR) and clinical pregnancy (CPR) rates were also analysed. During Phase 1 of the study we identified three variables most closely related to implantation: t3 (34-40 h), followed by cc2 (9-12 h) and t5 (45-55 h). Based on these results we elaborated an algorithm that classified embryos from A to D according to implantation potential. During Phase 2 of the study the algorithm was validated in a different group of patients that included 865 cycles and 1620 embryos transferred. In this phase of the study, embryos were categorized based on the algorithm and significant differences in IR were observed between the different categories ('A' 32%, 'B' 28%, 'C' 26%, 'D' 20% and 'E' 17%, P < 0.001). In addition we identified three quality criteria: direct cleavage from one to three cells, uneven blastomere size in second cell cycle and multinucleation in third cell cycle. The retrospective nature of the study limits its potential value, although the use of one database to generate the algorithm (embryos from this database were not selected by any morphokinetic criteria) and one database to validate it reinforces our conclusions. The elaboration of an algorithm based on a larger database derived from different (albeit related) clinics raises the possibility that such algorithms could be applied in different clinical settings.

Morphokinetic analysis of cleavage stage embryos and its relationship to aneuploidy in a retrospective time-lapse imaging study.

To analyze differences in morphokinetic parameters of chromosomally normal and aneuploid embryos utilizing time-lapse imaging and CGH microarray analysis. This retrospective cohort study included patients undergoing IVF treatment and preimplantation genetic diagnosis for sex selection. A total of 460 embryos cultured in incubators with time-lapse imaging system (EmbryoScope) were selected for biopsy on day 3 of development. Subsequently, CGH microarray analysis was performed for aneuploidy screening of 24 chromosomes. Kinetic parameters including time for appearance of second polar body (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPBf), time to division to 2(t2), 3(t3), 4(t4), 5(t5) cells, length of second and third cell cycle (CC2= t3 t2, CC3=t5-t3), synchrony of cell division from 2 to 4 cells (S2=t4-t3) and interval t5-t2 were analyzed to compare chromosomally normal and abnormal embryos. The mean time durations for tPNf, t2, t5, CC2, CC3, t5-t2 differed significantly between normal and abnormal embryos. Time-lapse imaging morphokinetics may play a role in early prediction of aneuploid embryos due to differences in kinetic behavior that may aid in improving clinical outcome.

Abstract 1983: Real-time imaging of exosomes dynamic of cross-talking and cell trafficking in 3D Gelfoam® histoculture

Abstract Exosomes are small membrane vesicles (30-100 nm) released by many types of cells during normal physiological processes and are thought to play an important role in cell-to-cell communication to promote tumor metastasis. Exosomes contain diffusible factors, such as cytokines, growth factors and extracellular matrix molecules and also mediate local and systemic cell communication through the horizontal transfer of information, such as mRNAs, microRNAs and proteins. However, the exosome dynamics and trafficking between cancer cells and tumor stromal cells in a metastatic niche are still unclear. In this study, in order to image the movement of cancer cell-derived exosomes, we developed a confocal time lapse imaging system to use green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes, expressed in breast cancer cells cultured on 3-dimentional Gelfoam® histoculture. We demonstrated that 3D Gelfoam® culture enabled imaging of structural cell-to-cell contact between neighboring cells and exosome behavior in real-time. This system makes it possible to monitor real-time exosome dynamics and cross-talking between cancer cells and other cells and to identify the mechanism of exosome release and uptake such as in a metastatic niche. Citation Format: Mako Yamamoto, Shinji Miwa, Yukihiko Hiroshima, Shuya Yano, Fuminari Uehara, Yasunori Matsumoto, Atsushi Suetsugu, Kimi Homma, Robert M. Hoffman. Real-time imaging of exosomes dynamic of cross-talking and cell trafficking in 3D Gelfoam® histoculture. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1983. doi:10.1158/1538-7445.AM2014-1983

Injection device built on a 3D printer for a lab-on-a-chip

OBJECTIVE: To investigate if nuclear status at 2 cell stage and reverse cleavage between 2 4-cell stage of transferred embryo, are related to implantation outcome. DESIGN: Retrospective study. MATERIALSANDMETHODS: After Intracytoplasmic sperm injection, the metaphase II oocytes were cultured in EmbryoScope (Unisense) timelapse system. Transferred embryos were retrospectively analyzed in EmbryoViewer database. Ninety-one transferred embryos from 52 women were analyzed. Forty-one implanted transferred embryos were from 25 patients and 50 non-implanted from 27 patients. The presence of multinucleation at 2-cell stage and reverse cleavage were compared between positive and negative implanted groups. Reverse cleavage (RC) was defined as a decrease in the number of cells during cytokinesis. Namely, cells in blastomere fused together to form a blastomere with lesser cells, and they cleaved again after that. This phenomenon was specifically observed with time-lapse imaging system, and noticed previously [1]. Implantation was assessed by ultrasound in 4 weeks after embryo transfer. Double embryo transfers with only one heart beat were excluded. Data were analyzed using c-test and P-value < 0.05 was considered significant. RESULTS: In the implanted group, 21(51.2%) embryos were observed withmononucleation at 2-cell stage, while 15 (36.6%) presented 1-cell multinucleation (C1), 5 (12.2%) presented both cells multinucleation(C2), and 5 (12.2%) presented RC. In the non-implanted group, 25(50%) embryos were observed with mononucleation at 2-cell stage, while 14 (28%) presented C1, 11 (22%) presented C2, and 6 (12%) presented RC. Comparisons of 2-cell stage mononucleation and multinucleation rates between implanted and non-implanted were not significant. The implantation rates of mononucleation, C1, and C2 were 45.7%, 51.7%, and 31.3% respectively. The implantation rates of RC and non-RC groups were 45.5 % and 45%. The differences between these implantation rates were not significant, either. CONCLUSION: Blastocysts from mono and multinucleated 2-cell embryos have equal chances of implantation success. Reverse cleavage is not a significant factor in unsuccessful implantation.

Multinucleation on 2-cell stage and reverse cleavage may not impact implantation outcomes: a time-lapse study

To investigate if nuclear status at 2 cell stage and reverse cleavage between 2∼4-cell stage of transferred embryo, are related to implantation outcome. Retrospective study. After Intracytoplasmic sperm injection, the metaphase II oocytes were cultured in EmbryoScope (Unisense) time-lapse system. Transferred embryos were retrospectively analyzed in EmbryoViewer database. Ninety-one transferred embryos from 52 women were analyzed. Forty-one implanted transferred embryos were from 25 patients and 50 non-implanted from 27 patients. The presence of multinucleation at 2-cell stage and reverse cleavage were compared between positive and negative implanted groups. Reverse cleavage (RC) was defined as a decrease in the number of cells during cytokinesis. Namely, cells in blastomere fused together to form a blastomere with lesser cells, and they cleaved again after that. This phenomenon was specifically observed with time-lapse imaging system, and noticed previously [1]. Implantation was assessed by ultrasound in 4 weeks after embryo transfer. Double embryo transfers with only one heart beat were excluded. Data were analyzed using χ2-test and P-value < 0.05 was considered significant. In the implanted group, 21(51.2%) embryos were observed with mononucleation at 2-cell stage, while 15 (36.6%) presented 1-cell multinucleation (C1), 5 (12.2%) presented both cells multinucleation(C2), and 5 (12.2%) presented RC. In the non-implanted group, 25(50%) embryos were observed with mononucleation at 2-cell stage, while 14 (28%) presented C1, 11 (22%) presented C2, and 6 (12%) presented RC. Comparisons of 2-cell stage mononucleation and multinucleation rates between implanted and non-implanted were not significant. The implantation rates of mononucleation, C1, and C2 were 45.7%, 51.7%, and 31.3% respectively. The implantation rates of RC and non-RC groups were 45.5 % and 45%. The differences between these implantation rates were not significant, either. Blastocysts from mono and multinucleated 2-cell embryos have equal chances of implantation success. Reverse cleavage is not a significant factor in unsuccessful implantation.

Time-lapse imaging system with shell-less culture chamber

We have developed a system for imaging whole chick embryos from embryonic day 1.5 (E1.5) to E4.5. Our system consists of a custom-made culture chamber, the top and bottom of which were heated and the inside was humidified. The system also has a fixed stage uplight fluorescent microscope, and long-working distance objective lenses were adopted. The albumen removed-yolk with the embryo in the dish was put in the chamber. It is of importance that we adopted long working distance lenses because the working distance of conventional objective lenses is too short for observation of the embryo in a humidified chamber. The objective lens we adopted has sufficient resolution to detect fluorescent protein expression at the single-cell level. Transparent glass heater set on the top of the chamber helps to reduce dew condensation; the bottom heater keeps the temperature inside the chamber, and the water bath surrounding the egg maintains humidity. This system was used to detect fluorescent protein expressing cells in embryos. We could successfully trace those cells for 17h in vivo. In conclusion, this system is useful for time-lapse analysis of fluorescent protein expression and distribution for a longer period of time.

Time-lapse imaging of cell cycle dynamics during development in living cardiomyocyte

Mammalian cardiomyocytes withdraw from the cell cycle shortly after birth, although it remains unclear how cardiomyocyte cell cycles behave during development. Compared to conventional immunohistochemistry in static observation, time-lapse imaging can reveal comprehensive data in hard-to-understand biological phenomenon. However, there are no reports of an established protocol of successful time-lapse imaging in mammalian heart. Thus, it is valuable to establish a time-lapse imaging system to enable the observation of cell cycle dynamics in living murine cardiomyocytes. This study sought to establish time-lapse imaging of murine heart to study cardiomyocyte cell cycle behavior. The Fucci (fluorescent ubiquitination-based cell cycle indicator) system can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei red, green and yellow, respectively, in living mammalian cells, and could therefore be useful to visualize the real-time cell cycle transitions in living murine heart. To establish a similar system for time-lapse imaging of murine heart, we first developed an ex vivo culture system, with the culture conditions determined in terms of sample state, serum concentration, and oxygen concentration. The optimal condition (slice culture, oxygen concentration 20%, serum concentration 10%) successfully mimicked physiological cardiomyocyte proliferation in vivo. Time-lapse imaging of cardiac slices from E11.5, E14.5, E18.5, and P1 Fucci-expressing transgenic mice revealed an elongated S/G2/M phase in cardiomyocytes during development. Our time-lapse imaging of murine heart revealed a gradual elongation of the S/G2/M phase during development in living cardiomyocytes.

Alpha-lipoic acid-loaded nanostructured lipid carrier: sustained release and biocompatibility to HaCaT cells in vitro

ALA-loaded nanostructured lipid carrier (ALA-NLC) was designed to improve physicochemical stability and water solubility, and promote sustained release of ALA as well as determine the biocompatibility of ALA-NLC. The ALA-NLC manufactured using hot high-pressure homogenization technique was investigated in terms of size, zeta potential, FTIR analysis and release behavior. In vitro cytotoxicity and biocompatibility were determined by incubating with HaCaT cells using the MTT assay, HE staining and Hoechst 33342 staining. Cell behavior and cellular division of HaCaT cells untreated and treated by ALA-NLC were investigated in real-time images gathered using time-lapse imaging system. The release investigation illustrated that only 6.9% of ALA released in 30 min from ALA-NLC formation, whereas it was 30.3% in free ALA system. ALA-NLC possessed a satisfactory release behavior of sustained release up to 72 h. It showed that ALA-NLC did not exert hazardous effect on HaCaT cells up to 81.9 mg/L without morphological alterations, revealing a satisfactory biocompatibility. Evidence was provided from time-lapse imaging system that cell behavior and cellular division of ALA-NLC treated HaCaT cells were in accordance with the control. These results of this investigation demonstrated that NLC encapsulated ALA formation (ALA-NLC) can improve stability, solubility and release of ALA; ALA-NLC was biocompatible to HaCaT cells.

Abstract 4997: Using three-dimensional (3D) culture systems to delineate the role of RANKL in metastatic prostate cancer.

Abstract Introduction and Objective: While prostate cancer (PCa) is curable at early diagnosis, metastatic PCa is terminal. Recent studies have shown that Receptor Activator of NF kappa-B Ligand, RANKL, markedly enhances bone metastasis (70-100%) by the LNCaP human PCa cell line in mice compared to neo controls (0%). Multivariable tumor and microenvironmental factors in vivo make it difficult to identify the specific cellular response induced by RANKL causing PCa cell homing and growth in bone. Current in vitro 2D cultures lack mechanical and biochemical properties mimicking the bone microenvironment. Our preliminary data showed integrin alpha2 is elevated in RANKL-over-expressing LNCaP cells. PCa interaction with the major bone matrix protein Col1 may contribute to the metastatic potential of LNCAP-RANKL cells. This study used 3D systems to explore the role of RANKL in PCa-bone matrix interactions. Methods: 3D cultures of RANKL-expressing (highly metastatic) or non-metastatic LNCaP cells were evaluated in 2D, suspension cultures, on type 1 collagen (Col1) coated polymeric meshes, embedded in Col1 gel or hydrogel mixed with Col1 for growth, morphology, motility, and gene expression. Time-dependent cell migration was assessed by a time-lapse imaging system. Results: LNCAP-RANKL cells and Neo-expressing controls grew as aggregates in all 3D cultures but not 2D cultures. Col1 promoted less compact and more widespread aggregation of LNCAP-RANKL cells in the 3D cultures, exhibiting more motile and directional migratory behavior. A molecular signature of 3D growth was generated and compared among different conditions. Consistent with 2D studies, 3D qRT-PCR data showed RANKL drove epithelial-to-mesenchymal transition (EMT), shown by increased expression of vimentin and N-cadherin but decreased E-cadherin. Decreased expression of androgen receptor and PSA was observed in RANKL-expressing PCa cells. Supporting the migration data, the presence of Col1 drove RANKL-expressing PCa cells to express higher levels of c-Met protooncogene, sensitizing PCa cell responses to the c-Met ligand, hepatocyte growth factor (HGF). Conclusions: 3D culture systems were established to study interactions between Col1 and metastatic or non-metastatic PCa cells. 3D culture systems supported PCa cell growth, while maintaining their classical markers, validating the reliability of our 3D systems. Bone metastatic PCa cells preferentially exhibited EMT, increased expression of α2 integrin, and c-Met protooncogene. These 3D systems will be used to assess liquid biopsy specimens harvested from human PCa patients to improve prognosis and therapeutic follow-up. Citation Format: Shabnam Ziaee, Shirly Sieh, Chia-Yi Chu, Ruoxiang Wang, Dietmar W. Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4997. doi:10.1158/1538-7445.AM2013-4997

Abstract B11: Using three-dimensional (3-D) culture systems to delineate the role of RANKL in metastatic prostate cancer

Abstract Introduction and Objective: While prostate cancer (PCa) is curable at early diagnosis, metastatic PCa is terminal. Recent studies have shown that Receptor Activator of NF kappa-B Ligand, RANKL, markedly enhances bone metastasis (70-100%) by the LNCaP human PCa cell line in mice compared to neo controls (0%). Multivariable tumor and microenvironmental factors in vivo make it difficult to identify the specific cellular response induced by RANKL causing PCa cell homing and growth in bone. Current in vitro 2D cultures lack mechanical and biochemical properties mimicking the bone microenvironment. Our preliminary data showed integrin alpha2 is elevated in RANKL-over-expressing LNCaP cells. PCa interaction with the major bone matrix protein Col1 may contribute to the metastatic potential of LNCAP-RANKL cells. This study used 3D systems to explore the role of RANKL in PCa-bone matrix interactions. Methods: 3D cultures of RANKL-expressing (highly metastatic) or non-metastatic LNCaP cells were evaluated in 2D, suspension cultures, on type 1 collagen (Col1) coated polymeric meshes, embedded in Col1 gel or hydrogel mixed with Col1 for growth, morphology, motility, and gene expression. Time-dependent cell migration was assessed by a time-lapse imaging system. Results: LNCAP-RANKL cells and Neo-expressing controls grew as aggregates in all 3D cultures but not 2D cultures. Col1 promoted less compact and more widespread aggregation of LNCAP-RANKL cells in the 3D cultures, exhibiting more motile and directional migratory behavior. A molecular signature of 3D growth was generated and compared among different conditions. Consistent with 2D studies, 3D qRT-PCR data showed RANKL drove epithelial-to-mesenchymal transition (EMT), shown by increased expression of vimentin and N-cadherin but decreased E-cadherin. Decreased expression of androgen receptor and PSA was observed in RANKL-expressing PCa cells. Supporting the migration data, the presence of Col1 drove RANKL-expressing PCa cells to express higher levels of c-Met protooncogene, sensitizing PCa cell responses to the c-Met ligand, hepatocyte growth factor (HGF). Conclusions: 3D culture systems were established to study interactions between Col1 and metastatic or non-metastatic PCa cells. 3D culture systems supported PCa cell growth, while maintaining their classical markers, validating the reliability of our 3D systems. Bone metastatic PCa cells preferentially exhibited EMT, increased expression of α2 integrin, and c-Met protooncogene. These 3D systems will be used to assess liquid biopsy specimens harvested from human PCa patients to improve prognosis and therapeutic follow-up. Citation Format: Shabnam Ziaee, Shirly Sieh, Chia-Yi Chu, Ruoxiang Wang, Dietmar Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3-D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B11.

Investigation of biocompatibility on nitrogen-doped a-C:H film coating scaffold surface in in-vivo and in-vitro tests

ABSTRACTIn this study, in order to investigate biocompatibility of nitrogen-doped hydrogenated amorphous carbon (a-C:H:N) film coating segmented polyurethane (SPU) scaffold fiber sheet (a-C:H:N-Scaffold) in in-vitro test, mouse fibroblasts (NIH 3T3) cells were grown on the a-C:H:N-Scaffold. The cell behavior was monitored by time-lapse imaging system. Additionally, the a-C:H:N-Scaffold was implanted at partial aorta descendens of a goat for 35 days. The surface morphology, composition, and wettability of the a-C:H:N-scaffold was estimated by Scanning Electron Microscope (SEM), X-ray photoelectron spectrometer (XPS), and contact angle measurement. In in-vitro test, it was observed that a-C:H:N film coating had a facilitatory effect on cell motility and cell growth. In in-vivo test, it was observed that the a-C:H:N-Scaffold surface was uniformly covered by neointima. The a-C:H:N-Scaffold surface had no thrombus formation as an inflammatory reaction and it was shown that the a-C:H:N film coating had a good blood compatibility. These results suggest that a-C:H:N film coating has good cytocompatibility and blood compatibility and it is a promising approach for improvement of biocompatibility of biomaterial surfaces.

High Efficient Time Lapse Imaging System for Cyclic Stretched Cells

High Efficient Time Lapse Imaging System for Cyclic Stretched Cells

Time-lapse imaging and the ideal time for embryo cleavage on the third day of development: preliminary results

Time-lapse imaging and the ideal time for embryo cleavage on the third day of development: preliminary results