Cell cultures represent a standard in vitro model in biomedical research. Without the need of using laboratory animals, they allow for the complex study of cellular mechanisms, both under physiological, and experimental conditions. Their application ranges from the study of metabolic routes in basic research, to production of recombinant proteins in the biotech or pharmaceutical industries.The techniques of working with cell cultures have been well established and at MQA, we have succeeded in establishing and standardizing a number of in vitro methodologies, which are now offered through our molecular services division.
Mycoplasma Detection Services
We provide testing of eukaryotic cell lines for the presence of all mycoplasma species from the culture media in a very short time. Mycoplasmas are the smallest and simplest prokaryotes. They are causal agents of numerous diseases of animals and humans and may also cause annoying contaminations in eukaryotic cell cultures. The presence of mycoplasmas in cellular lines may significantly influence the results of experiments because mycoplasmas may affect: 1) the cell growth rate which induces morphological changes which induces chromosomal aberrations, 2) the amino and nucleic acids’ metabolism inducing cell transformation, 3) molecular transport through the cell membrane.
For Human Vaccines and Biologicals Compliant with 21 CFR 610.30
Multi-Media Direct Culture Method with Fluorochrome Staining Assay
This test consists of a multi-media Direct Culture method using three different mycoplasma media formulations combined with an Indicator Cell Culture procedure. The Indicator Cell Culture procedure with DNA fluorochrome assay enhances the detection of non-cultivable mycoplasma species. This is the best test for detecting mycoplasma contamination in cell cultures and is a GMP service.
DNA Fluorochrome Staining Assay with Indicator Cell Line
This DNA fluorochrome assay with inoculation on an indicator cell line enhances the level of assay sensitivity by reducing background from genetically unstable cell lines (e.g. hybridomas, etc.) and amplifies the titer of mycoplasma contaminants. Developed for the detection of mycoplasma contamination in cell cultures and related samples, this test provides reliable and timely results.
PCR Based Detection Assay
Incomparably the fastest and easiest of all the tests done at MQA. In this assay, cellular particles of the cell culture supernatant are isolated by centrifugation and the DNA is separated and purified. Aliquots are used in PCR reactions with a primer mixture for the amplification of sequences which occur in the conserved 16S rDNA regions of almost all mycoplasma species listed so far in publicly available sequence databases. After contamination, a 504 – 519 bp DNA fragment (depending on the mycoplasma species) is visible in the ethidium bromide stained agarose gel. As an internal control, we coamplify a ca. 975 bp DNA fragment in parallel to verify successful PCR amplification regarding sensitivity and absence of Taq polymerase inhibitors.
Other Mycoplasma Services Include
- Reagent Standards
- Mycoplasma DNA for Assay Development
- Positive and Negative Control Slides for in-house development
- MQA specially coated slides for your lab to use at your convenience. Simply add your cell culture sample, fix, and dry. Return the (hazard-free) slides to our laboratory for DNA fluorochrome staining and diagnosis.
Primary Cell Line Generation
Primary cells are cells straight from the tissue with no passages. A primary cell culture may be composed of mixtures of cell types. Frequently the some of the cells may survive without proliferating and will therefore be lost in the increasing population of those which are able to multiply in the conditions supplied in vitro.
- Cells from explants may sometimes be converted to cell lines by passage. These may continue to proliferate for a number of cell generations.
- MQA also offers cells for use of primary fibroblasts as feeder layers for the growth of some embryonic stem cell types. At MQA our scientists can prepare primary cell lines from most tissues using our standard protocols. If you would like to implement a custom project, we would be happy to discuss your project specifications with you.
Testing by MTT, XTT, or any customized approach, such as Alamar blue, is based on the ability of metabolically active cells to convert special dyes into measurable flurophores such as yellow tetrazolium salt, XTT, into orange formasan. Complex rinsing is thus avoided, which is necessary, for example, in the older MTT method. This guarantees high accuracy of the XTT test and allows for fast processing of the data obtained. The bioassay may also be used to establish relative, cytotoxicity of agents within various chemical classes. We can establish baseline data for predicting the toxicity of related novel agents by comparing such baseline data with known in-vivo toxicity. The assay is simple to perform since the indicator is water soluble, thus eliminating the washing/fixing and extraction steps required in other commonly used cell proliferation assays.
Authentification by Isozyme Assay
One of the major problems in cell culturing is the misidentification or cross-contamination of authentic continuous cell lines. Confirmation of purity and identity of cell cultures is a necessary step in the production of biotherapeutics. Working with several cell lines in the same facility can result in cross contamination while mislabeling of cultures can cause misidentification. The method is based on the isoelectric separation of a specific set of intracellular enzymes which can be used to distinguish between human, murine, or other mammalian cell lines.
Stable Cell Line Expression
For either over or knock-down by shRNA. Stable cell lines with specific gene over-expression or knock-down are very helpful in gene function analysis, target discovery, target validation, assay development, and compound screening. MQA’s team of experienced scientists will work with you to establish the system to generate your particular cell line for your specific application. Since long-term suppression using pol III shRNAs can be problematic especially for studies of genes that are critical to cell proliferation and differentiation, we provide not only constitutive but also conditional knock-down.
MQA Service Will Provide You With
- Your choice of a constitutive or inducible promoter, a tag, a selection marker and a fluorescent marker . Either you provide target templates or we design, synthesize, or obtain it from a cDNA collection.
- Clone your gene or shRNA into our lentiviral vector.
- Generate lentivirus.
- Transduce the cell line of interest.
- Select the stably transduced, high expression cells.
- Validate the genomic integration of the target by genomic PCR.
- Validate the high-expression clone by Western Blot (if applicable).
- Produce two cryogenic preserved vials of stable cells and a final report.
Why Use MQA Cell Culture Services
- Safe-to-use (self-inactivating) lentiviral particles can deliver your gene into a wide range of cell lines including non-dividing, primary or stem cells.
- Engineered in-house lentiviral vector for highly efficient gene integration into cell genome.
- Our lentivectors and standard C-type retroviral vectors have the strongest promoter for the highest protein expression in mammalian cells.
- You can choose to have an inducible* or constitutive promoter.
- Our experts have years of experience in lentiviral cloning and expression.
- Fast turnaround time.
- The best price and the best quality in its class
Other Cell Culture Based Services
- Cell viability (cytotoxicity)
- Cell proliferation
- Cell adhesion
- Cell migration
- Cell invasion
- Cell shape change
- Soft Agar