Biomedical Engineering Reference
In-Depth Information
35.2.5
Safety Tests
Before the beginning of cell culture, patients' sera (4 mL) was
tested for hepatitis B surface (HBs) antigen (chemiluminescent
immunoassay [CLIA]), HBs antibody (CLIA), hepatitis B core (HBc)
antigen (CLIA), hepatitis C virus (HCV) antibody (RIA solid phase),
human immunodeficiency virus (HIV) antigen and HIV antibody
(enzyme-linked immunosorbent assays [ELISAs]), syphilis serology
(rapidplasmaregain[RPR]),syphilisserology(
Treponema pallidum
hemagglutinationassay [TPHA]), humanT-celllymphotrophic virus
(HTLV)-1 antibody (CLIA), parvovirus B19 DNA (polymerase chain
reaction [PCR]), and mycoplasma antibody (ELISA). The patients
were negative for all tests. Peripheral blood (3 mL) was also tested
for white blood cell, red blood cell, and platelet counts; hemoglobin,
hematocrit,meancorpuscularhemoglobin(MCV),andmeancorpus-
cular hemoglobin concentration (MCHC).
Before cell culture, DMEM with 10% autologous serum (1 mL)
underwent two safety checks. To test for contamination by bacte-
ria andfungi, the medium wasspread onto horsebloodagar (Nissui
Pharmaceutical) and the medium was also tested for mycoplasma.
Mycoplasma DNA was extracted from the prepared medium using
phenol/chloroform/isoamyl alcohol (PCI) (Sigma). Equal volumes
of PCI were added to the medium (600
μ
L) and centrifuged at
15,000 revolutions/min for 5 minutes at room temperature. The
supernatant was then mixed with 400
μ
L ice-cold 100% propan-
2-ol (Wako) and centrifuged at 15,000 revolutions/min for 10
minutes. The pellet was then rinsed with 400
μ
L ice-cold 70%
ethanol (Wako) and centrifuged at 15,000 revolutions/min for 5
minutes. The final pellet was dried for 3 minutes and then dis-
solved in 20
μ
L distilled water for PCR. The PCR mix (25
μ
L) con-
tained 2.5
Lof
10 mM of each primer (F1: s'-ACACCATGGGAGYTGGTAAT-3'; R1: s'-
CTTCWTCGACTTYCAGACCCAAGGCAT-3'), and 0.1
μ
L10
×
buffer, 2
μ
L of each 10 mM dNTP, 0.5
μ
μ
μ
L
Taq-DNA polymerase (Takara). Thirty-five cycles were run with the
following conditions: 94
◦
C for 1 minute (denaturation), 55
◦
Cfor
30 seconds (annealing), and 72
◦
C for 30 seconds (polymerization).
Amplified DNA was separated on a 1.5% agarose gel and soaked in
Tris/acetate/ethylenediaminetetraacetic acid (EDTA) (TAE) buffer
L of 5 units/
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