| Part 1:Production and infectivity of PERVs by the co-culture system of porcine hepatocytes and MSCs with galactosylated chitosan nanofiber scaffoldsAIM:To detect the production and infectivity of PERVs released by the co-culture system of porcine hepatocytes and MSCs with galactosylated chitosan nanofiber scaffolds.METHODS:The primary porcine hepatocytes or the mixed suspension of hepatocytes and MSCs during passages 3 to 5 (2:1) was cultured for 7 days in the 6 well cell culture plates with or without the galactosylated chitosan nanofiber scaffolds (defined as Hepatocytes (Hep) group, nanofiber scaffold (Nano) group, co-culture (Co) group and nanofiber scaffold & co-culture (Nano-Co) group, respectively). The culture media were collected daily to detect the reverse transcriptase (RT) activity with RT activity assay kits, the PERV RNA by RT-PCR and real-time PCR with the PERV specific primers, and the PERV protein gag p30 with Western blotting. In vitro infectivity of the supernatant was tested by incubating HEK293 cells.RESULTS:Two peaks of PERV expression were found at 10H and day 2 and followed by a regular decline in Hep group and Nano group. Presence of the virus lasted five days in the former group and six days in the later group. But in Co group and Nano-Co group, the PERV release gradually increased after 2 days to the peaks in Day 6. By Comparision, the virus levels in the Nano-Co group after two days were obviously higher than that in the Hep group or than that in the Nano-Co group at 10H.. No HEK293 cell was infected in vitro by the supernatant, although some microchimerism was observed in Co group and Nano-Co group.CONCLUSION:PERV release was confirmed in the co-culture system of porcine hepatocytes and MSCs, and when the cells were cultured with galactosylated chitosan nanofiber scaffolds the viruses increased gradually over time with a significant higher production after 2 days than simple primary hepatocytes or its level at 10H in Nano-Co group. However, no obvious in vitro infectivity was found although microchimerism appeared in the last few days. The results suggest it reasonable that the co-culture system of porcine hepatocytes and MSCs with galactosylated chitosan nanofiber scaffolds be used within two days after inoculation.Part 2:The influence of the plasma component separators with different membrane pore sizes on the transfer of PERVs in the novel BALAIM:To detect the transfer of PERVs across the plasma component separators with different membrane pore sizes, and to find a plasma component separator with suitable pore size for the novel BAL.METHODS:The mixed suspension of hepatocytes and MSCs during passages 3 to 5 (2:1) was perfused into the novel multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds to establish a new BAL system composed of three circuits. The BAL systems were divided into four groups according to the membrane pore size of the plasma component separators, that is, l0nm group,20nm group,30nm group and 35nm group. The media flow was initiated 4 hours after the cells were seeded and lasted 48 hours. The culture media in each circuit were collected at regular intervals to detect the reverse transcriptase (RT) activity with RT activity assay kits, the PERV RNA by RT-PCR and real-time PCR with the PERV specific primers, and the PERV protein gag p30 with Western blotting. In vitro infectivity of the supernatant was tested by incubating HEK293 cells.RESULTS:Except the transmission of PERV RNA through the plasma component separators and plasmafilters at 48 hours in 30nm group and 35nm group, no PERV RNA, PERV capsid protein, and RT activity was detected in the supernatant in the external circuit of all groups at any time point, suggesting no PERV particles across the plasma component separators with different membrane pore sizes. In addition, no HEK293 cell was infected in vitro by the supernatant, although some microchimerism was observed with the media from the Circuit 3.CONCLUSION:No transfer of infectious PERVs across the plasma component separators with different membrane pore sizes was found during the proposed duration of the treatment, but PERV RNA was able to pass through the plasma component separators with membrane pore sizes>20nm. Therefore, it may be safer in the virology to apply the plasma component separators with mambrane pore sizes≤20nm.Part 3:Study on the virological safety of the novel BAL in therapeutic applicationAIM:To evaluate the virological safety of the novel BAL in therapeutic application.METHODS:Five dogs of galactosamine-induced ALF were treated for 3 hours with the new BAL based on the new multi-layer radial-flow bioreactor containing galactosylated chitosan nanofiber scaffolds and co-system of porcine hepatocytes and MSCs in the second day after models established. The plasma in each circuit and the whole blood of the canines were collected before, during and after the treatment, and the dogs were sacrificed to retrieve the tissue of hearts, livers, spleen, lung and kidneys. These fuild samples were used to detect the PERV RNA, DNA, and RT activity, and the immunohistochemisty and western blot were performed to find the PERVs capsid protein gag p30 in the tissue. Furthermore, the HEK293 cells were incubated by the plasma to determine their In vitro infectivity.RESULTS:PERV RNA, DNA and RT activity found in the plasma of Circuit 3 revealed PERV particles released by the co-cultured cells existing in Circuit 3. No PERV RNA, DNA, RT activity and anti-PERV antibody was detected in he other plasma including that from the other circuits and the whole blood of the dogs, and no HEK293 cells was infected by all the plasma samples in vitro. In addition, the results of all PERV-related analysis in the PBMCs and the tissue were negative, suggesting no transfer of PERVs across the plasma component separators and no experimental dogs infected.CONCLUSION:No evidence of transmission of PERVs into the ALF canines with the treatment of the new BAL was observed, which proved the reliable virological safety of the new BAL. |
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