SEVENTH INTERCOUNTRY MEETING OF DIRECTORS OF POLIOVIRUS LABORATORY 24 26 AUGUST 2003 AMMAN , JORDAN

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SEVENTH INTERCOUNTRY MEETING OF DIRECTORS OF
POLIOVIRUS LABORATORY24 - 26 AUGUST 2003
AMMAN , JORDANImplementation of QC in cell
culture , VACSERA
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Introduction

• Place
• Our cell culture unit was established 3 years
  ago.
• Space of the lab is adequate and completely
  separated from other laboratories.
• The lab is well - equipped , regularly calibrated
  cleaned and well- maintained.

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• Materials
• Media (ready made) from highly specified company
  with certificates.
• FBS from highly specified company with
  certificates.
• All the used reagents are analytical grades .
• Establishment of our cell bank started with RD
  and L20B strains from WHO- GSL.

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• Staff
• The cell culture considered as specified
  activities , it requires a staff of considerable
  experience.
• We began with long term experience technician
  and then trained other technicians.
• All documents , sops and maintenance records are
  written and maintained.

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QUALITY CONTROL

• Q.C means conformance to acceptable requirements.
• Q.C for C.C begins from setting up of cells from
  liquid nitrogen till freezing.
• Q.C of cell cultures is a vital process and we
  must be attention for 3 characteristics
• PURITY.
• AUTHENTICITY.
• STABILITY.

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1-PURITY

• The cells must be pure which means, free from
  contaminants
• bacteria.
• fungi.
• virus.
• Mycoplasma.
• routine checks for bacterial, fungi and
  Mycoplasma are relatively easy to establish and
  will provide confidence in the quality of cell
  culture results.

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DETECTION OF MYCOPLASMA

• We used to do PCR test to detect Mycoplasma in
  VACSERA C.C unit.
• Now we already implement this test as a routine
  test in our PCR unit.
• The next slide will show this.

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Result of PCR Mycoplasma Test
LANE 1 L20B 1st PRIMER SET LANE 2 RD
1st PRIMER SET LANE 3 VE C 1st
PRIMER SET
LANE 4 L20B 2nd PRIMER SET LANE 5 RD
2nd PRIMER SET LANE 6 VE C 2nd
PRIMER SET LANE 7 MARKER LANE 8 - VE C
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2-AUTHENTICITY

• All our cells are obtained from Global Polio
  Laboratory Network.
• To avoid cross contamination only one cell line
  is handled at a time in BSC.
• Between two types of cell lines the work area
  must be cleaned and disinfected.

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3- STABILITY

• To avoid the variation in genetic or phenotypic
  characteristics all cell lines are replaced after
  a maximum of 15 sequential passages so
  sensitivity test is implemented through this
  period.

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• We used to do this test by using OPV TYPE 2
  already calibrated in Vacsera Q.C department.
• Now after receiving the reference strains and the
  protocol from NIBSC we did our working stock of
  Q.C standards and calibrated with NIBSC
  standards.

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Calibration of working Vacsera standards
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• We did the sensitivity test at the early stages
  of cell subculture (passage 2), the midway
  (passage 7) and the end of subculture (passage
  14) to insure the reliability of results
  during this period .

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CRITERIA OF THE TEST

• 1- One hand.
• 2- Using Sabin like 2 strain.
• 3- Three successive days.
• 4-Titer is the same or 0.5 of the expected
  reference value.
• 5-If no reproducibility the cell line should be
  replaced from liquid nitrogen.

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Sensitivity test results during period of sample
inoculation
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Handling of cell culture in sample inoculation

• We put in our consideration that
• Handling of one cell line at a time.
• Temperature of incubator 36ºC.
• PH of G.M and M.M is 7.2 7.4.
• Seeding level depend on condition of cells,
  spacing and date of previous seeding.
• counting or splitting ratio (1/3 1/4) both are
  the same in our lab .

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AUG / 2003
THANKS