ISCBI guidance tables

Tables and Figures for the ISCBI Guidance Document Feb 08


Table 1.  Proposed Testing for hESC Master Cell Banks

Test Specification Examples of Test Methods Typical Test Result Pass Specification Release Criteria
Identity Matches parent cell line

Short Tandem Repeat (STR) Testing

Human Leukocyte Antigen (HLA) Testing

Shares all alleles of parent cell line. Passes test result specification
Bacteria/Fungi No contamination detected Innocultation of mcirobiological culture media to detect growth of bacteria and fungi * No culturable bacterial or fungal organisms detected ** Passes test result specification
Mycoplasma No contamination detected Direct culture in broth and agar and indirect test using indicator culture/DNA stain * No culturable mycoplasma detected ** Passes test result specification
Karyotype Report karyotype from a specified number of metaphase analyses (see section 5.2 of this guidance) Perform G-band analysis on 20 metaphase spreads. Further analysis may be performed using FISH. Single karyotype in all cells analysed. No alternative karyotypes at or above 5% of metaphase 'spreads' in the cell preparation (see sectcion 5.2 of this guidance) Passes test result specification
Post-Thaw Recovery Viable colonies recovered (quantified efficiency of recovery of each bank/lot should be given) Test for the ability to recover viable hESC colonies. Colonies recovered that are representative of the original cell line as demonstrated in other quality control and characterisation data *** Passes test result specification
Growth Characteristics Report value Growth characteristics under standard cell culture conditions. Determine doubling time. Growth rate estimate presented -
Cell Antigen Expression High proportion of cells (typically >70%) positive for each marker Flow cytymoetry for a range of hESC markers (e.g. SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-1 negative) Data presented -
Cell Gene Expression Report data available Gene expression profiling using DNA microarray or Q-PCR analysis. Analyse for expression of core hESC genes as well as markers of differentiated cell types. Data presented if available -
Genetic Stability Report data available Analysis of multiple Single Nucleotide Polymorphisms, SKY or Comparative Genome Hybridization by DNA microarray method. Data presentd if available -


*Cell differentiation capacity can be determined during characterization studies performed on distribution lots/banks.  These studies should be performed on cells passaged beyond the distribution lot passage level e.g. 20 passages
**Use appropriate controls to assist in validation of result, sensitivity determination and false negatives due to inhibition of assays. Sensitivity should be known and validated for hESC samples in the hands of the testing laboratory.
***Bank to establish a justified specification that may be dependent on the needs of customers. This is a developing area in which banks should maintain knowledge of current developments and best practice. Coordination between banks is vital to continue to improve viability of preserved cells supplied to bank users.


 
Table 2 Mycoplasma Detection Methods

Technique/Test Method Comments
Selective broth and agar subculture/industry standard methdos are published in national pharmacopoeia
  • Generally considered the most sensitive method for detecting culturable mycoplasma and acholeplasma species.
  • Lon incubation period are required with final results not complete for serveral weeks.
DNA stain (Hoechst or DAPI) of innoculated Vero cells/industry standard methods are published in national pharmacopoeia
  • Can isolate mycoplasma strains that do not grow in broth/agar method.
  • Simple to carry out providing uv-epiflourescent microscope is available with high magnification.
  • Results available within a few days give good sensitivity obtained using culture methods.
  • Rapid direct staining can be performed but may suffer from cell and bacterial aretefacts leading to ambiguous results.
PCR/variety of methdos based on amplification of mycoplasma DNA e.g., 16S rRNA, 23S rRNA, rpoB, ITS. Representative reference include Eldering et al., 2004; van Kuppeveld et al., 1994; Sung et al., 2006 and Upphoff and Drexler, 2005
  • Detects non-culturable organisms.
  • Useful for screening cultures due to easy sample preparation and rapid productio of test results (within hours).
  • May be very senstitive but some methods can lead to false positives (important to validate the specific method used for sensitivity and specificity).
  • PCR assays are also subject to limited snesitivity due to small sample volume and inhibition of the PCR reaction by sample components. Sensitivity can be address by use of diluted DNA controls and sample spiking with positive controls (Upphoff and Drezler, 2005).
Detection of mycoplasma specific enzymes/commercially available kits
  • MycoTect (Invitrogen): detects a wide range of mycyoplasma species based on levels of the mycoplasma-specific enzyme adenoside phosphorylase.
  • MycoAlery (Cambrex): reported to be capable of detecting down to 20 cuf/mL of several mycoplasma species.

 

Table 3. Examples of STR Genetic Alleles Represented in Commercially Available Kits

Gene Locus Name Applied Biosystems Promega
Cofiler (USA) ProfilerPlus (USA) Identifiler (UK) SGM+ (UK) Power Plex 16 (UK, USA)
Amelogenin + + + + +
D3S1358 + + + + +
D16S539 + - + + +
TH01 + - + + +
CSF1PO - - + - +
TPOX + - + - +
D7S820 + + + - +
D13S317 - + + - +
D21S11 - + + + +
D18S51 - + + + +
D8S1179 - + + + +
FGA - + + + +
vWA - + + + +
D5S818 - + + - +
D2S1338 - - + + -
D19S433 - - + + -
Penta D - - - - +
Penta E - - - - +

 

Table 4 List of Biological Reagents used in Stem Cell Derivation and culture and associated microbiological hazards

Reagent Source Potential Contaminants
Fetal calf serum Bovine fetuses Bovine viruses e.g. bovine viral diaffhea virus, bovine polymoma virus (numerous serum free media available but may still contain materials of animal original)
Trypsin Porcine pancreas Porcine virsues (risk of contamination reduced by using recombinant trypsin produced in recombinant plants*)
Bacterial enzymes such as Collagenase Bacterial cultures of Clostridium spp. Spores and organisms from the original culture*
Growth factors Animal/human tissues and cells Viral contamination depending on the species of origin*

*NB Altering the source of a biological reagent using recombinant DNA approaches may eliminate certain hazards but if the new source of material is still of biological in origin (e.g. recombinant organisms) then there may still be materials used in its preparation that represent a risk of contamination, however, this will generally not be a significant issue for materials used for research purposes.


 
Table 5. The Karyological Analysis of hESC lines: Recommended Terms, Standard Method and Procdures for Investigation of Abnormal Results and Reporting of Data.

Standard G-band Analysis

10 metaphases analysed

20 metaphases counted

Procedure for investigation of clonal abnormal findings Clonal chromosome abnormalities should be confirmed in a second stage later passage culture, to allow further interpretation of their significance
Procedure for investigation of abnomalities observed in single cells Single cell abnormalities (e.g. aneuploids, structural rearrangements) will require further investigation in some cases to exclude mosaicism, depending on the chromosome involved
Aneuploidy of chromosomes 1, 8, 12, 14, 17, 20 and X (including unbalanced rearrangements) A total of 30 G-banded cells counted from initial culture. Examine 100 interphases using FISH with appropriate probe of a follow up later passage culture and 30 G-banded cells
Other aneuploidy and structural abnormalities A total of G-banded 30 cells counted from initial culture
Minimum quality score

ISCN 400 band level is the minimum level of G-banding analysis necessary, although effort should be made to analyse cells of ISCN 500 band level and above.

The method used to score the banding is at the laboratories discretion. An example of a banding scoring system can be found in the UK's Association of Clinical Cytogenetics Best Practice Guidelines (2007)

www.cytogenetics.org.uk/prof_standards/acc_general_bp_mar2007_1.01.pdf

Substandard analysis In the analysis at the ISCN 400 band level cannot be achieved, the analysis can proceed as normal but should be reported with the caveat that it is a "substandard analysis" and may need to be repeated
Report

The result should include:

  • The karyotype designation using current correct ISCN (2005) nomenclature where practicable
  • The types of analysis used (e.g. karyotype, FISH, CGH, special types of banding, etc)
  • The average banding level achieved
  • Single cells with aneuploidy or structural abnormalities involving chromosomes 1, 8, 12, 14, 17, 20 or X (this list is evolving) should be reported, even after extended analysis, as it is necessary to analyse a second later passage culture to fully interpret the abnormality
Definitions (adapted from ACC General Best Practice Guidelines, 2007)

Analyse - to count a metaphase and compare every chromosome, band for band, with its hmoologue and to verify the banding pattern of the X and Y-chromosomes in male karyotypes.

Count - to enumerate the total number of chromosomes in any given petaphase in such a way that gross structural abnormalities would be identified, or in FISH analysis to enumerate the number of signals in an interphase nucleus.

Score - to check for the presence or absence of abnormalities in a cell or metaphase without full anaylsis.

Clone - a cell population originally derived from a single cell

Such cells will have an identical chromosome constitution. A clone is said to exist if three cells have lost the same chromosome, or two cells contain the same extra or structurally rearranged chromosome.

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