Webshop log in View product details. Description: Cell counting chamber Purpose: For accurate analysis of semen samples. Chamber depth: 20 Number of chambers: 2. Chamber depth: 20 Number of chambers: 4.
Width mm : For correct analysis of semen samples Motility and concentration of spermatozoa are part of the assessments made to determine semen quality. Semen analysis with MicroCell Watch this short movie for a demonstration on how to use the MicroCell for semen analysis. Advanced design ensures accuracy The hydrophobic, non-vented chamber design eliminates sample leakage, resulting in greater sample stability and enhanced safety.
Support Documents. Therefore, we decided to evaluate our newly developed transgene expression vector MiP as an alternative shuttle system for the delivery of the reprogramming factors We were able to reprogram numerous somatic human somatic cells fibroblasts, PBMCs, keratinocytes and renal epithelial cells and mouse cell lines C57Bl6 via a single electroporation.
Compared to other established non-integrating reprogramming methods, the 4-in-1 CoMiP has multiple advantages. It is color-labeled, time-efficient, inexpensive and easy-to-use, thus providing an attractive alternative for use in regenerative medicine. In addition, because of the engineered intronic region of the polycistronic 4-in-1 CoMiP plasmid, the overall size of our construct was significantly reduced, which in turn increased the transfection efficiency in different somatic cell types.
Moreover, the smaller size allowed us to include a RFP marker gene to track the transfection efficiency of our target cell lines. We hypothesize that the higher exogenous transgene expression rate mediated by the intronic sequence and the codon optimization of the 4-in-1 CoMiP plasmid are sufficient to reactivate the endogenous circuit of pluripotency within the first few days, whereas more persistent transgene expression would require other EBNA1- and OriP-based episomal vectors.
Interestingly, we observed a significantly reduced time course for the induction of pluripotency using the 4-in-1 CoMiP, which allowed us to select the first iPSC colonies 12 days after the initial transfection. Furthermore, the 4-in-1 CoMiP reprogramming protocol is compatible to fully chemically defined culture conditions using E7 and E8 media, which may prove to be important for future clinical applications of iPSCs.
By contrast, the EBNA1 and OriP sequences used in the Yamanaka episomal reprogramming system are derived from the human pathogenic EBV and thus carry the risk of possibly altering the innate immune response-triggered gene expression of the subsequently derived iPSCs and may limit their clinical applications 6.
The use of DNA-based reprogramming methods always carries the potential risk of random genomic integration of the transfected DNA into the host cell genome Thus, the transfection of fewer plasmids 1 plasmid in the case of the 4-in-1 CoMiP vs. Compared to other single reprogramming plasmids described so far, our 4-in-1 CoMiP protocol is more efficient and is highly reproducible in a broad variety of somatic cell types 8 , 32 , 35 , Keratinocytes, however, were relatively difficult to reprogram, resulting in only a few iPSC colonies, probably due to the change from specialized keratinocyte media into more chemically-defined conditions of the E7 or E8 media.
By contrast, the reason for the enhanced reprogramming process of the renal epithelial cells may be their propensity for mesenchymal-to-epithelial transition and their higher expression levels of E-Cadherin 37 , 38 , 39 , Therefore, we believe that the reprogramming efficiencies of the different constructs used in this study also depend on the cell type used for the reprogramming experiment 6 , Aside from the aforementioned DNA-based reprogramming methods, there are three other commonly used integration-free techniques for inducing pluripotency: protein addition, mRNA transfection and Sendai virus infection.
The disadvantages of the protein reprogramming technique are that it is expensive, inefficient and slow 21 , Using modified mRNA for the induction of pluripotency is an elegant and unique approach that guarantees derivation of integration-free iPSCs without any further screening experiments. However, this method is laborious and requires a repetitive series of daily mRNA transfections for up to 14 days and the pre-treatment of the initial cell types with the expensive interferon alpha antagonist B18R This molecule is crucial for enhancing cell survival during the series of mRNA transfections and for efficient reprogramming Another limitation of this technique is its dependency on the feeder cell and conditioned media, which brings an extra risk of potentially transmitting undetected human pathogens A recent publication addressed one of the aforementioned disadvantages of mRNA based reprogramming.
However, further validation is required to establish how robust and reproducible this particular method is. By comparison, the Sendai virus is perhaps the most efficient integration-free reprogramming method currently available.
However, it is also the most expensive method and requires more stringent biosafety containment measures and a separate tissue culture room. The persistence of residual viral material requires an extended period of tissue culture time 10 to 20 passages to establish virus-free iPSC lines for further downstream analysis and differentiation experiments Finally, another recently published approach used small-molecule compounds to reprogram mouse somatic cells However, the efficiency of this technique is also quite low and the study must be reproduced using human somatic cells in order to be of broader clinical interest.
In summary, the 4-in-1 CoMiP is an effective alternative reprogramming method for deriving integration-free iPSCs from various donor tissue sources, including PBMCs under chemically defined conditions without using animal-derived products. Compared to other DNA-based reprogramming methods like the minicircle or the Yamanaka three individual plasmids system, the CoMiP construct is faster and less expensive for inducing pluripotency in a somatic cell type Supplementary Fig.
Furthermore, the cost effectiveness of our new reprograming technique should be emphasized because of the current impetus for major research institutions to generate large-scale iPSC banks. Hence this CoMiP construct will allow novice and inexperienced researchers alike to easily obtain bona fide iPSCs within 14 days. Aside from being the fastest reprogramming method, the 4-in-1 CoMiP plasmid does not result in intermediate iPSCs and, due to its color label, the entire reprogramming process is easy to monitor.
With all these superior qualities, we believe that this new technology is of special interest given the great potential of iPSCs in regenerative medicine. The 1.
The positive colonies were then selected for sequencing to determine the insertion orientation in the resultant 4-in-1 CoMiP plasmid. However, only the backward insertion was able to grow in this experiment design.
This provided the final version of 4-in-1 CoMiP vector. The minicircle plasmids were produced as described previously 6 , Reprogramming using the minicircle technique was performed as previously described with an optimized minicircle backbone 4-in-1 CoMiC and some changes in the reprogramming procedure The following paragraph describes the fibroblast transfection procedure, which was identical for all reprogramming plasmids used in this study.
Subsequently, these cells were equally distributed onto one or two Matrigel-coated plates 5. On the following morning day 1 , the media was changed to fibroblast media with the addition of 0. On day 3, the media was changed to Essential 7 media supplemented with 0.
Between day 6 4-in-1 CoMiP and day 20 4-in-1 minicircle CoMiC, Yamanaka episomal plasmids the first iPSC-like colonies appeared, after which we switched the culture conditions to Essential 8 medium and hypoxic conditions. Around days 12—30, the iPSC colonies were large enough for manual selection under the microscope. After 5—7 days, the individual colonies became big enough to dissociate into single cells using 0. Takahashi, K. Induction of pluripotent stem cells from fibroblast cultures.
Nat Protoc 2, — Yu, J. Induced pluripotent stem cell lines derived from human somatic cells. Science , — Bock, C. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell , — Lee, J. Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis. Arthritis Res Ther 16, R41 Article Google Scholar.
Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Jia, F. A nonviral minicircle vector for deriving human iPS cells. Nat Methods 7, — Warren, L. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.
Cell Stem Cell 7, — Chou, B. Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.
Cell Res 21, — Okita, K. A more efficient method to generate integration-free human iPS cells. Nat Methods 8, — Mandal, P. Reprogramming human fibroblasts to pluripotency using modified mRNA. Nat Protoc 8, — Narsinh, K. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat Protoc 6, 78—88 Diecke, S. Second generation codon optimized minicircle CoMiC for nonviral reprogramming of human adult fibroblasts.
Methods in Mol Biol , 1—13 Kay, M. A robust system for production of minicircle DNA vectors. Nat Biotechnol 28, — Lu, J. A mini-intronic plasmid MIP : a novel robust transgene expression vector in vivo and in vitro. Mol Ther 21, — Tiemann, U.
Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells. Cytometry A 79, — Aggen, D. Gene Ther 19, — The extragenic spacer length between the 5' and 3' ends of the transgene expression cassette affects transgene silencing from plasmid-based vectors. Mol Ther 20, — Warlich, E.
Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming. Mol Ther 19, — Papp, B. Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape. Chen, G. Chemically defined conditions for human iPSC derivation and culture. Activation of innate immunity is required for efficient nuclear reprogramming. Stetson, D. Immunity 24, 93— Burridge, P.
Chemically defined generation of human cardiomyocytes. Nat Methods 11, — Vitrolife Academy provides a wide range of webinars within the field of IVF. Our webinars are recorded and you can find our recorded webinars here, to watch at your convenience. Working together with Vitrolife means having a partner that understands the overall challenges involved in running an IVF clinic. Learn more about the IVF Journey from a patient perspective and how Vitrolife products can support your treatment.
Here you will be able to walk through the IVF clinic of the future displaying different solutions and products. From Diagnostics to Pregnancy. We guide you through the different steps. Save time by placing your orders online. If you have not yet activated an online account and want to be able to place orders online, access CoA's, see your invoices, previous orders and much more - it's easy to request an account.
Our products come with a comprehensive package insert including instructions for use. This is however not the only thing we offer to secure the best usage. You can also educate yourself through the multitude of supporting documents and instructional movies to support optimal product use and facilitate different ways of learning. Vitrolife's dedication to quality control is ultimately reflected in the quality of our products and services.
Our commitment is founded on deep knowledge and genuine devotion to your success in IVF. Please find more information about working at Vitrolife and about how to apply for an available position.
0コメント