[Study on the setup of a new technique of tissue and cell culture for vitreous fluid of vitrectomy].
OBJECTIVE
To setup a new technique of tissue and cell culture for vitreous aspirates.


METHODS
Experiment study. Specimens used for supporting new culture technique were selected based on random digit table. Thirty cases with rhegmatogenous retinal detachment (RRD) and forty-eight with proliferative diabetic retinopathy (PDR), which undergoing primary pars plana vitrectomy, were selected randomly and included in the study. After being antiphase stained with fluorescein-natrium (0.5%) and digested with hyaluronidase (10(5) U/L) combined with collagenase I (10(6) U/L) for removing vitreous gel, sediment of vitreous fluid after centrifugation were inoculated into standard culturing bottle with which polylysine (0.01%) was pre-set. The bottle which contained F12 medium with 30% fetal bovine serum was placed upside down for 24 hours and consecutively upside for 6 days. During which, F12 medium was replaced once in half volume, and cell growth along the edge of sedimentary membrane was observed at time of the 3rd and the 6th day after upside culture.


RESULTS
Under condition of pre-setting by polylysine (0.01%) and being placed upside down for 24 hours, pieces from vitreous fluids could adhere to the bottom of bottle in a way of semi-xerosis with adherence rate of 100% (78/78). No bacteria, fungus and mycoplasma contamination was found within 7 days. Antiphase stained with fluorescein-natrium (0.5%) and digested with hyaluronidase (10(5) U/L) combined with collagenase I (10(6) U/L) for 30 minutes, vitreous gel in 78 specimens could be digested (78/78). Cell emigration could be found in edge area of some pieces of vitreous fluid and cell growth as well as proliferation was shown. In 30 specimens of RRD, cell growth rate were 43.33% (13/30). In 48 specimens of PDR, cell growth rate were 37.50% (18/48). Concerning PDR phase V (PDR-V), cell growth rate reach 41.67% (10/24).


CONCLUSIONS
Enzymolysis with upside down and semi-xerosis could ensure good adherence of membrane, moreover, no contamination and obvious cell growth could be found within short-term culture. These suggested that a new technique for judging viability of cell from proliferative membrane and predicting recurrent risk after surgery of proliferative ocular fundus disease could be expected.
Li
Gen-lin
GL
Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, Beijing 100730, China. ligenglin@263.net
Liu
Yue-yue
YY
Zhang
Xiang
X
Zhang
Cheng-yue
CY
Wang
Liang-hai
LH
D000293:Adolescent; D000328:Adult; D000368:Aged; D001826:Body Fluids; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D046509:Tissue Culture Techniques / Q000379:methods*; D014821:Vitrectomy; D014822:Vitreous Body / Q000166:cytology*; D055815:Young Adult