Method for Screening of Agents for the Prevention of Hepatitis C Virus Infection with Cell Culture Tool
The invention relates to an improved method of screening of anti-HCV agents that may have an efficacy for prevention of hepatitis C virus. The method involves the isolation and cryopreservation of HCV-infected hepatocytes from multiple infected individuals. The isolated and cryopreserved hepatocytes are stored in a cryopreservation bank made up of HCV-infected hepatocytes representing the different genotypes of HCV. These stored hepatocytes then are co-cultured in a culture medium with uninfected hepatocytes, and anti-HCV screening of the hepatocytes is done by subjecting HCV infected hepatocytes and uninfected hepatocytes in parallel to the actions of different anti-HCV compounds at various concentrations. An effective anti-HCV agent will lead to prevention of increase in concentration of HCV content of uninfected cells in the co-culture.
1 . A method for co-culturing HCV-infected and uninfected human hepatocytes to screen for agents that prevent the transmission of HCV, the method comprising
retrieving HCV infected and uninfected hepatocyte cells from a cryopreservation bank;
thawing the cells in a warm water bath;
suspending the cells in a medium;
culturing the cells in a plate with multiple inner wells;
interconnecting the inner wells with a fluid medium;
providing one or more anti HCV agents; and
quantifying the HCV content of the co-culture by quantification of HCV RNA.
2 . The method of claim 1 , wherein the anti-HCV agent is added to the fluid medium used for interconnecting the wells.
3 . The method of claim 1 , wherein multiple anti-HCV agents are used.
4 . The method of claim 1 , wherein the multiple anti-HCV agents are added at different concentrations.
5 . The method of claim 1 , wherein the co-cultured uninfected hepatocytes and infected hepatocytes are connected by a fluid medium.
6 . The method of claim 5 , wherein the fluid medium used for interconnecting the wells is DMEM/F12 medium containing 10% of fetal calf serum (FCS), insulin (10 ug/mL), and dexamethasone (100 nM).
7 . The method of claim 1 , wherein the quantification of HCV RNA is performed by RT-PCR.
8 . The method of claim 1 , wherein the temperature of the warm water bath is approximately 37° C.
9 . The method of claim 1 , wherein the suspending medium is DMEM/F12 medium containing 10% of fetal calf serum (FCS), insulin (10 ug/mL), and dexamethasone (100 nM).
10 . The method of claim 1 , wherein the cell plate comprises a collagen-coated plate.
11 . The method of claim 1 , wherein the cell plate has six inner wells.
12 . The method of claim 11 , wherein three wells are used for culturing infected hepatocytes.
13 . The method of claim 11 , wherein three wells are used for culturing uninfected hepatocytes.
14 . The method of claim 1 , different genotypes of HCV infected cells are co-cultured with uninfected hepatocytes in multiple cell plates.
15 . A method for co-culturing infected and uninfected hepatocytes in a medium to evaluate a level of HCV infection through quantification of HCV production by infected hepatocytes, the method comprising:
extraction of total RNA of uninfected hepatocytes;
quantification of RNA of uninfected hepatocytes; and
quantification of an HCV titer of the medium and infected hepatocytes by quantification of RNA of HCV.
16 . The method of claim 15 , wherein the quantification of RNA of HCV is performed by RT-PCR.