Objectives: The efficient acquisition and purification of fibroblasts as ideal seed cells are very important. For optimization of the isolation and culture of human foreskin fibroblasts (HFF), we compared the improved tissue culture method (ITCM) and the enzyme digestion method (EDM).
Methods: In ITCM, the skin tissue was digested with 0.1% Type II collagenase overnight at 4 ℃, the epidermis was separated from the dermis and digested again with 0.25% trypsin at room temperature for 15 min, and then the tissue block was attached to the culture dish. In EDM, the skin tissue was digested with 0.25% trypsin overnight at 4 ℃, the epidermis was separated from the dermis and digested with 0.1% Type II collagenase overnight at 4 ℃, the tissue block was filtered and squeezed together with the enzyme mixture, the filter was rinsed with medium containing fetal bovine serum, and the cell suspension was cultured. Both ITCM and EDM used 2 digestion enzymes, but the order, digestion time, and temperature of the 2 enzymes were different. The final inoculations of ITCM and EDM in the dishes for subsequent culture were tissue blocks and cell suspensions, respectively. In this study, HFF cells were isolated and cultured with ITCM and EDM, and the cell morphology was observed from Passage 0 to Passage 3 in the ITCM and EDM groups. The cell purity was identified by staining for vimentin, CD68, and Pan-keratin. The growth curves of Passage 3 were plotted to compare the proliferation ability of the 2 groups. Passage 3 HFF cells in the ITCM and EDM groups were irradiated with medium-wave ultraviolet (UVB) at an energy value of 120 mJ/cm2 to establish a light damage model. The experiments were grouped into an UVB group and a control group (Control) according to the presence or absence of UVB irradiation. Platelet-poor plasma (PPP) was extracted by secondary centrifugation, and the HFF cells of ITCM and EDM groups were cultured in groups using complete medium containing different concentrations (0, 2.5%, 5.0%, and 10.0%) of PPP, and the proliferation of damaged cells was detected by cell counting kit-8 after 24 h of PPP incubation.
Results: A large number of HFF could be observed in the ITCM group up to day 3, which was less affected by impurities; the observation of HFF morphology in the EDM group was affected by more impurities. By day 9, cells in both ITCM and EDM groups could be passaged; HFF isolated and cultured in vitro by the 2 methods showed long spindle-shaped, swirling growth. The positive rates of vimentin in the ITCM and EDM groups when HFF cells were cultured up to Passage 2 were significantly different [(97.36±0.76)% vs (99.4±0.56)%, P<0.01)]. The positive rates of CD68 were also significantly different [(70.8±0.46)% vs (78.37±0.75)%, P<0.01]. The expressions of pan-keratin in the ITCM group and the EDM group were positive and negative, respectively. There was no difference in vimentin and pan-keratin staining results between the ITCM group and the EDM group when HFF were cultured to Passage 3. The positive rates of CD68 between the ITCM group and the EDM group were significantly different [(74.73±1.37)% vs (85.27±2.63)%, P<0.001]. The proliferative capacity of HFF cells in Passage 3 was significantly higher in the EDM group than that in the ITCM group (P<0.05). After UVB (120 mJ/cm2) irradiation, HFFs procured by the 2 isolation methods showed damage. The damage repair test demonstrated that the 2.5% PPP+UVB irradiation group showed significantly higher repair competence than the other groups (all P<0.05).
Conclusions: In contrast with HFFs isolated via ITCM, HFF cells isolated by EDM have a faster purification rate and a stronger proliferative capacity. Therapy with PPP can moderately repair UVB-induced damage to HFFs. The results provide a theoretical basis for clinical treatment studies in the future.
目的: 作为理想的种子细胞，成纤维细胞的高效获取及纯化尤为重要。本研究通过比较改良组织块培养法(improved tissue culture method，ITCM)及酶消化培养法(enzyme digestion method，EDM)分离培养人包皮成纤维细胞(human foreskin fibroblasts，HFF)，以探讨两种方法的优缺点。方法: ITCM是将剪碎的皮肤组织置0.1% II型胶原酶中于4 ℃下消化过夜，分离表皮与真皮，对真皮组织采用0.25%胰酶于室温下再次消化15 min，待组织块贴壁于培养皿后进行培养。EDM是将剪碎的皮肤组织置0.25%胰酶中于4 ℃下消化过夜，分离表皮与真皮，将真皮组织再次于4 ℃下用0.1% II型胶原酶消化过夜，然后将组织块与酶的混合物一起过滤、挤压，用含有胎牛血清的培养基冲洗滤网，对得到的细胞悬液进行培养。ITCM与EDM均采用两种酶进行消化，但两种酶的使用顺序、消化时间及温度不同，最终接种于培养皿中进行后续培养的分别是组织块和细胞悬液。本研究利用ITCM和EDM分离、培养HFF，观察ITCM组和EDM组HFF的原代至第3代(Passage 0~Passage 3，P0~P3)的细胞形态；染色波形蛋白、CD68和角蛋白以鉴定细胞纯度；绘制P3生长曲线对比两组细胞的增殖能力。采取120 mJ/cm2的中波紫外线(medium-wave ultraviolet，UVB)照射P3 ITCM组和EDM组HFF，建立光损伤模型。根据有无照射紫外线，实验分为UVB组和未照射对照组(Control)。采取二次离心法提取贫血小板血浆(platelet-poor plasma，PPP)，用含不同浓度(0，2.5%，5.0%及10.0%)PPP的完全培养基对ITCM组和EDM组的HFF进行分组培养，加入PPP 24 h后用CCK-8试剂盒检测损伤细胞的增殖情况。结果: ITCM组培养至第3天时可观察到大量HFF，受杂质影响小；EDM组HFF形态的观察受到较多杂质影响；第9天时，两组细胞均可传代；两种方法体外分离培养的HFF均呈长梭形、旋涡状生长。HFF培养至P2时ITCM组和EDM组波形蛋白的阳性率比较差异有统计学意义[(97.36±0.76)% vs (99.4±0.56)%，P<0.01]，CD68的阳性率比较差异有统计学意义[(70.8±0.46)% vs (78.37±0.75)%，P<0.01]，pan-keratin的表达分别为阳性和阴性。HFF培养至P3时ITCM组与EDM组比较，波形蛋白和角蛋白染色结果差异无统计学意义(均P>0.05)，CD68的阳性率比较差异有统计学意义[(74.73±1.37)% vs (85.27±2.63)%，P<0.001]。EDM组P3 HFF的增殖能力显著高于ITCM组(P<0.05)。经UVB(120 mJ/cm2)照射后，两种方法获取的HFF出现光损伤改变；2.5% PPP组修复损伤细胞的能力显著高于其余浓度组(P<0.05或P<0.001)。结论: 与ITCM法比较，EDM法获得的HFF具有纯化速度快、增殖能力强的优点；PPP对HFF的紫外线损伤具有修复作用。实验结果可为后续临床治疗研究提供理论依据。.
Keywords: cell identification; enzyme digestion method; human foreskin fibroblasts; improved tissue culture method.