What is the reason for heat-inactivation prior to usage?
Heat-inactivation (heating to 56°C for 45 min) inactivates proteases of the complement system. This could be important for cells which are difficult to culture, or cells which will be used to prepare or assay viruses, used in cytotoxicity assays or other systems where complement may have an unwanted influence. On the other hand, heat inactivation may deplete serum growth factors.
Which sera are routinely used at the DSMZ as growth supplements?
The DSMZ cell lines were all propagated in FBS/FCS (fetal bovine/calf serum) or HS (horse serum). With one exception (PB-1, ACC-no. 241) we use only heat-inactivated serum.
Which cell culture media are used at the DSMZ?
The media we use are currently sourced from Invitrogen. This, however, should not be taken as an endorsement. All media contain L-glutamine.
Why are passage numbers omitted from the cell line data sheets?
We believe that passage numbers are unreliable criteria for ageing cell lines. Given that "passage" is defined as the trypsination of a culture and its subsequent re-seeding, passage-numbering arbitrarily depends on the split ratio used by the individual cell culturist. Thus, if operator A splits 1:2, and B 1:4, they will end up with two different passage numbers ("B" half of "A"), even though both cell cultures will have been growing for the same time period.
How should I prepare samples for mycoplasma testing?
1) Cryopreserved cell cultures should be cultured for circa 2 weeks prior to testing to ensure a sufficient mycoplasma titer. Thus, we recommend submission of samples from growing cultures to minimize chargeable costs for shipment and cell culture.
2) For the same reason, cell lines must be cultured without antibiotics for at least two weeks prior to submission.
3) We recommend submission of samples taken on Monday's (conditioned medium from a three days culture or older) and immediate shipment. Do not send the samples later than Wednesday to ensure same-week delivery.
4) Work in DSMZ laboratories is only permitted for cell cultures categorized up to and including risk group 2.
Suspension cell cultures:
For suspension cell lines, stand culture flasks vertically allowing cells to settle for about 30 minutes prior to removal of 2 ml of the supernatant and transfer to two sterile tubes.
Adherent cell cultures:
Grow the cultures almost to confluence and remove 2 ml supernatant. Transfer the supernatant to two sterile reaction tubes.
These procedures should yield optimum numbers of cells to detect mycoplasma adhering to the eukaryotic cells to facilitate detection of adherent mycoplasma.
How should I send samples for mycoplasma testing?
Seal the reaction tubes with Parafilm and wrap carefully. Send the samples unchilled by conventional mail or courier chosen to ensure same-week delivery.
If you prefer to send frozen cultures, add sufficient dry ice (about 4 kg) and send by courier for same-week delivery.
Address package to:
Dr. Cord Uphoff
DSMZ - Department of Human and Animal Cell Lines
Mascheroder Weg 1b
D-38120 Braunschweig, Germany
How is the mycoplasma PCR assay performed?
We first isolate the cellular particles present in 1 ml culture supernatant by centrifugation, and wash with PBS. Sample DNA is separated and purified using Wizard DNA Clean-Up system (Promega) and aliquots used as template in PCR reactions with primers for 16S rDNA regions of mycoplasma species predominantly found in cell cultures. In the event of contamination, a 504 - 519 bp DNA fragment (depending on the mycoplasma species) is visible in the ethidium bromide-stained agarose gel. As internal control, we amplify a 975 bp DNA fragment added in a limiting dilution in a parallel PCR reaction to confirm a successful PCR amplification. Positive, negative, and water controls are also included.
For comparison of the PCR method with other mycoplasma detection assays, please refer to the following publication:
Uphoff CC, Drexler HG: Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell Dev Biol Anim 38: 79-85 (2002).
How sensitive is the PCR assay?
The determination of the sensitivity of a mycoplasma detection assay is problematical because an objective reference is necessary to quantitate mycoplasmas. The counting of colony forming units (cfu) is inexact because mycoplasmas often grow as clumps and one colony might be seeded from multiple mycoplasmas. Thus, we have cultivated pure cultures of mycoplasmas, isolated and quantitated the DNA, and performed PCR reactions with a dilution series of the DNA.
The following calculation is an approximation to determine the sensitivity:
- Amount of DNA after column preparation: 300 ng in 20 μl
- The genome size is assumed to be 1000 kb. 300 ng DNA corresponds to ca. 3 x 10E8 mycoplasmas (1 ng DNA / 10E6 mycoplasmas).
- 1 μl of a 1:100.000 dilution of the original DNA solution may be detected by the PCR reaction --> 300 ng / 20 μl : 100,000 = 150 fg/μl
- 150 fg correspond to a DNA amount of about 150 mycoplasma genomes (1 fg / mycoplasma genome)
How long does the elimination procedure take?
Depending on the antibiotic used, the eradication procedure takes at least four to five weeks.
How is mycoplasma elimination performed at the DSMZ?
We apply different antibiotics to eliminate the mycoplasmas. After treatment, the cell cultures are further cultured for at least two weeks without antibiotics. Two independent detection assays are then performed (PCR and microbiological culture). When the cultures are free from mycoplasma, the cells are expanded and five ampoules are frozen as stocks. Four ampoules are sent to the client and one stored for about 6 months in case of recurrence contamination. For further details on the elimination procedure and the antibiotics applied, please refer to the following publication:
Uphoff CC, Drexler HG: Comparative antibiotic eradication of mycoplasma infections from continuous cell lines. In Vitro Cell Dev Biol Anim 38: 86-89 (2002).
Which viruses are detected in human and primate cell lines?
We have established PCR assays for the detection of the human pathogenic viruses including Hepatitis B viruses (HBV), Hepatitis C viruses (HCV), Human immunodeficiency virus type 1 (HIV1), Human immunodeficiency virus type 2 (HIV2), and Human T-cell leukemia virus types I and II (HTLV-I/-II). To determine the transformation of B cells by Epstein Barr virus, we also established an assay for the detection of EBV, which is categorized as risk group 2.
Additionally, we are able to detect Human herpes virus type 8 (HHV-8) and Transfusion transmitted virus (TTV).
How should I prepare and send samples for virus detection?
Human pathogenic viruses exhibit greater genomic diversity than bacterial contaminants. The human pathogenic viruses consist of DNA and RNA viruses and have different infective characteristics. Thus, different sample materials and procedures are used for the analyses. The following list shows the sample material matching the different virus types:
HIV-1, HIV-2, HTLV-I/-II, EBV: genomic DNA
HCV, EBV: total RNA or cDNA
HBV: DNA from cell culture supernatant
When frozen cultures are sent we will thaw and culture the cells for several days to collect the samples. This additional labour incurs a surcharge. Cryopreserved cultures should be sent on dry ice by courier.
If you send a growing culture, we will immediately collect the samples depending on the assays to be performed. This is the easiest and least expensive way to perform the assays. The culture vessels should be filled with medium and may be sent by conventional mail or courier despatched no later than on Wednesday's.
DNA and/or RNA/cDNA may also be sent. This material should be sent refrigerated or as precipitate at room temperature. Supernatants for HBV detection maybe sent at room temperature.
How much material should I send?
For viable cells, about 5x10E6 cells are sufficient for the extractions of DNA and RNA (see submission form page 2 for detailed information on how to prepare and how to send the material).
For nucleic acid preparations, the DNA of about 2x10E6 cells is sufficient for our needs. If the detection of hepatitis C virus is ordered, please send an extra preparation of total RNA also from about 2x10E6 cells.
What does the report look like?
The report consists of a description of the method, the result of the analysis, and a photo of the ethidium bromide stained gel.