The Scale-up Culture Of Encapsulated Hepatocytes

Aims:To investigate the effects of rotate speed and cell density on the growth of encapsulated HepA and HepG₂.Methods:Alginate-chitosan encapsulated HepA and HepG₂,were randomly divided into two groups:encapsulated HepA as treated group and encapsulated HepG₂ as control.Both groups were randomly divided into eight little groups according to rotate speed of 0RPM/2RPM/3RPM/4RPM and encapsulated cell density of 1.0X10⁶/2.0X10⁶ respectively.The initially total cell number was 1.0X10⁷.The size and number of spheroids were caculated. The expressions of Ki67 were also assessed by Elivision plus immunohistochemical technique.Results:The average number of spheroids increased from 1.1-4.5 at 2nd day to 3.2-13.1 at 8th day rapidly.The average maximal diameter of spheroids was 23.8-68.7um at 2nd day and mushroomed to 44.9-128.9um at 8th day.Then both number and size of spheroids all grew slowly.The average number was 5.4-18.5 and the average maximal diameter was 50.6-183.4um at 14th day.At any time,the strongly positive express rates of Ki67 in 0RPM group were lowest(d₂ 0-5%,d₇ 40-45%,d₁₂10-20%),but those in 4RPM group were highest(d₂ 65-70%,d₇ 100%,d₁₂75-80%),The weakly positive rates in 0RPM group were highest(d2 70-75%,d₇ 45-50%,d₁₂75-80%),but those in 4RPM group were lowest(d₂ 30-35%,d₇0%,d₁₂20-25%).The cells all not proliferated distinctly at first day in each group,then they all grew rapidly and came to a head at the 8th day.the total cell number reached(4.02-4.79),(17.69-19.79), (22.95-25.83)and(35.11-36.87)×10⁷ in 0RPM,2RPM,3RPM and 4RPM group respectively.Then cell growth rate slackened,they arrived at(5.87-6.42),(23.86-24.88),(30.18-30.89)and(43.06-43.92)×10⁷ at the 14th day.Conclutions:The number and the size of spheroid,the strongly positive express rates of Ki67,the proliferation rate were enhanced with increasing rotate speed in each group(P=0.00), but they all had no relation with encapsulated cell density(P＞0.05).The results suggested no marked difference in the strongly and weakly positive express rates of Ki67,the number and the size of spheroids and the proliferation rate in each group at any time(P＞0.05). Aims:To investigate the effects of rotate speed and cell density on the function of encapsulated HepA and HepG₂.Methods:Alginate-chitosan encapsulated HepA and HepG₂ were randomly divided into 48 groups according to the same rule as that of the first part.Albumin synthesis were assessed by Elisa,Urea synthesis was analysed using auto biochemcial analyzer,Diazepam clearance was detected by high performance liquid chromatography. Semi-quantitative reverse transcription PCR was used to detect the expression of CYP3A4,CYP2E1 and Cx32 in each group.Results:The albumin synthesis increased from 0.14-0.71pg/cell/h at the 2nd day to 0.60-3.50pg/cell/h at the 10th day rapidly.Then they attenuated quickly,no albumin could be detected at the 14th day.The urea synthesis of HepA and HepG₂ groups mushroomed from 0.09-0.23pg/cell/h and 0.16-0.62pg/cell/h at the 2nd day to 0.46-4.92pg/cell/h and 0.60-6.68pg/cell/h at the 10th day rapidly.But there were only 0.13-2.51pg/cell/h and 0.19-3.72pg/cell/h at the 14th day respectively.Diazepam clearance of HepA and HepG₂ groups increased from 0.41-2.42pg/cell/h and 0.12-0.69pg/cell/h at the 2nd day to 1.67-11.87pg/cell/h and 0.71-5.08pg/cell/h at the 10th day separately.But only 0.62-5.77pg/cell/h and 0.24-1.72pg/cell/h were found at the 12th day respectively.They could not be detected at the 14th day.The Ct ratio of CYP3A4,CYP2E1 and Cx32 were all less than one at any time in 0RPM group.In 2RPM/3RPM/4RPM encapsulated HepA groups,the Ct ratio of CYP3A4 were all more than one anytime.Those of CYP2E1 and Cx32 were all more than one at the 7th day.At the 12th day, the Ct ratio of Cx32 could remain more than one only in 4RPM group,but those of CYP2E1 were all more than one in 3RPM and 4RPM groups.In 2RPM/3RPM/4RPM encapsulated HepG₂ groups,the Ct ratio of Cx32 were all less than one at any time.But those of CYP2E1 and CYP3A4 were all more than one at the 2nd and the 7th day.At 12th day, the Ct ratio of CYP2E1 were all more than one only in 3RPM and 4RPM groups,but those of CYP3A4 were all less than one.Conclutions:Albumin synthesis,urea synthesis,diazepam clearance,the expression of CYP3A4,CYP2E1 and Cx32 were improved with increasing rotate speed in each group(P=0),but they all had no relation with encapsulated cell density(P＞0.05).In encapsulated HepA group,the results suggest marked differences in urea synthesis,diazepam clearance, the expression of CYP3A4 and Cx32(P＜0.05),although there were no diversity in albumin synthesis and the expression of CYP2E 1 at any time(P =0.84).