In vitro human lymphocyte proliferation assay under different extraction conditions and doses of two types of test samples / 中国组织工程研究

ABSTRACT

BACKGROUND:In vitro lymphocyte proliferation test is often used to detect the potential immunogenicity of medical devices,but no detailed extraction conditions and dose are given in the relevant standards. OBJECTIVE:To investigate the effects of different extraction conditions of the test product and different doses of the extract on in vitro human lymphocyte proliferation,and to consider the factors that need to be considered when selecting test conditions for in vitro lymphocyte proliferation test. METHODS:In the experiment,the homogenous bone repair material and heparin-modified intraocular lens were divided into the following 12 groups:(1)Experimental group 1:24-hour complete medium(RPMI modified medium containing 10%fetal bovine serum)extract of 200 μL + lymphocyte suspension of 50 μL;(2)negative control group 1:24-hour complete medium 200 μL + lymphocyte suspension 50 μL;(3)experimental group 2:24-hour complete medium extract 100 μL + lymphocyte suspension 100 μL;(4)negative control group 2:24-hour complete medium 100 μL + lymphocyte suspension 100 μL;(5)experimental group 3:72-hour RPMI modified medium extract(addition of 10%fetal bovine serum before experiment)200 μL + lymphocyte suspension 50 μL;(6)negative control group 3:72-hour RPMI modified medium(addition of 10%fetal bovine serum before experiment)200 μL + lymphocyte suspension 50 μL;(7)experimental group 4:72-hour RPMI modified medium extract(addition of 10%fetal bovine serum before experiment)100 μL + lymphocyte suspension 100 μL;(8)negative control group 4:72-hour RPMI modified medium(addition of 10%fetal bovine serum before experiment)100 μL + lymphocyte suspension 100 μL;(9)positive control group 1:complete medium containing 10 μg/mL plant hemagglutinin-M 200 μL + lymphocyte suspension 50 μL;(10)positive control group 2:complete medium containing 10 μg/mL plant hemagglutinin-M 100 μL + lymphocyte suspension 100 μL;(11)blank control group 1:250 μL complete medium;(12)control group 2:200 μL complete medium.After 3 days of culture,the proliferation of lymphocytes was detected by CCK-8 assay. RESULTS AND CONCLUSION:(1)Under different test conditions,the extracts of the allogeneic bone repair material could enhance the activity of human lymphocytes.Under the condition of 72-hour leaching in RPMI modified medium and the volume ratio of leaching solution and lymphocyte suspension was 4:1,the most significant effect was observed.Heparin-modified intraocular lens extract also had obvious inhibitory effect on lymphocyte activity under this condition;its inhibitory effect on lymphocyte activity may be related to the heparin in the extract.However,the activity of lymphocytes was slightly enhanced by heparin-modified intraocular lens extract under the experimental conditions of complete medium extraction for 24 hours and the volume ratio of extract to lymphocyte suspension was 4:1.(2)Under different extraction conditions and doses,the results of in vitro lymphocyte proliferation test may be quite different.The selection of test conditions should be combined with the clinical application of the product,and the inherent characteristics of the product should also be considered.