CAJ | 학술논문

目的：利用微尺度技术（Micro-scale technologies）建立人脂肪干细胞（hADSCs）体外三维培养平台，观察空间堆叠状态对种植后的hADSCs增殖和凋亡的影响。方法利用光刻技术在硅晶元表面生成直径60μm、80μm、100μm和150μm的微柱状结构，经聚二甲基硅氧烷（PDMS）倒模、固化生成同规格的微孔阵列（Micro-well arrays），加装PDMS环形外壁后组成微型反应器，测量、分析此实验平台的加工误差率；第3代hADSCs配制成1×105 cells/mL、6×104 cells/mL、3×104 cells/mL三种浓度单细胞悬液，并各取200μL分别接种于纯平PDMS微型反应器及直径60μm、80μm、100μm和150μm微孔微型反应器内，选择种植后24 h、72 h、120 h和168 h为采样点，对各观察组细胞分布状况进行形态学描述、细胞计数及活/死细胞检测。结果各孔径PDMS微孔阵列的微孔口径精度误差率均不超过0.2%；档hADSCs以6×104 cells/mL浓度接种时，细胞基本可以实现在150μm微孔内呈单层平铺、100μm微孔内呈2层堆叠、80μm微孔内呈3层堆叠；各微孔内细胞数在7 d内均无明显的数量变化，60μm组微孔内细胞数最少且微孔外平面残留细胞最多（P<0.05）；60μm组微孔内凋亡细胞比例高于其他组（P<0.05）。结论微加工倒模制作的PDMS微孔阵列培养平台误差率低，可满足高精度设计需要；hADSCs短时间（7 d）接种于微孔内处于增殖静止状态，能够保持三维堆叠状态稳定，便于进行堆叠状态下的细胞学早期研究；hADSCs在不同口径微孔内可以实现不同的堆叠层数，其凋亡比率与堆叠层数有关，堆叠层数少的细胞凋亡比例低于堆叠层数多者。
목적：이용미척도기술（Micro-scale technologies）건립인지방간세포（hADSCs）체외삼유배양평태，관찰공간퇴첩상태대충식후적hADSCs증식화조망적영향。방법이용광각기술재규정원표면생성직경60μm、80μm、100μm화150μm적미주상결구，경취이갑기규양완（PDMS）도모、고화생성동규격적미공진렬（Micro-well arrays），가장PDMS배형외벽후조성미형반응기，측량、분석차실험평태적가공오차솔；제3대hADSCs배제성1×105 cells/mL、6×104 cells/mL、3×104 cells/mL삼충농도단세포현액，병각취200μL분별접충우순평PDMS미형반응기급직경60μm、80μm、100μm화150μm미공미형반응기내，선택충식후24 h、72 h、120 h화168 h위채양점，대각관찰조세포분포상황진행형태학묘술、세포계수급활/사세포검측。결과각공경PDMS미공진렬적미공구경정도오차솔균불초과0.2%；당hADSCs이6×104 cells/mL농도접충시，세포기본가이실현재150μm미공내정단층평포、100μm미공내정2층퇴첩、80μm미공내정3층퇴첩；각미공내세포수재7 d내균무명현적수량변화，60μm조미공내세포수최소차미공외평면잔류세포최다（P<0.05）；60μm조미공내조망세포비례고우기타조（P<0.05）。결론미가공도모제작적PDMS미공진렬배양평태오차솔저，가만족고정도설계수요；hADSCs단시간（7 d）접충우미공내처우증식정지상태，능구보지삼유퇴첩상태은정，편우진행퇴첩상태하적세포학조기연구；hADSCs재불동구경미공내가이실현불동적퇴첩층수，기조망비솔여퇴첩층수유관，퇴첩층수소적세포조망비례저우퇴첩층수다자。
Objective To establish a three-dimensional cultivation platform for human adipose derived stem cells (hADSCs) by utilizing micro-scale technology, and to explore the influence of three-dimensional stacking condition to cell proliferation and apoptosis. Methods Micro-columnar structure were generated by micro-scale technology on silicon master with diameter of 60 μm, 80 μm, 100 μm and 150 μm respectively. The same specification micro-well arrays were acquired by reversing mould aperture using polydimethylsiloxane (PDMS), which equipped with PDMS ring on the outer wall to constitute micro-reactor. The error rate of this platform was analyzed. Then the third generation of human adipose-derived stem cells (hADSCs) were selected and mixed into single cell suspension mixture with three kinds of concentration, including 3×104 cells/mL, 6×104 cells/mL and 1×105 cells/mL. And 200μL cell suspension was injected into micro-reactor to establish three-dimensional culture models with different stacked layers. Then 24 hours, 72 hours, 120 hours and 168 hours were choosed as sample collection time point. The morphology description of cell distribution in each group was analyzed with scanning electron microscopy, the arrangement inside the microwells and the cell distribution outside the microwells were described, and the live/dead cells were counted and tested in each aperture and micro hole by using laser scanning confocal microscope. Results The error rate of diameter and pore diameter were no more than 0.2%. When hADSCs were planted by the density of 6 ×104 cells/mL, the stacking layer condition can be acquired. The number of cell within microwells was at static condition no more than 7 days. The 60 μm group had the highest number of out-of-well cell (P<0.05) and lowest 05). The highest apoptosis ratio was also observed in the 60 μm group (P<0.05). Conclusion The application of microscale technique can acquire extremely low error rate, which can satisfy the requirements of high precision design. The static proliferation condition of planted hADSCs can be maintained within microwells, which can stabilize the condition of cells and be designed as a perfect three dimention model for cell research at early stage of plant. The stackable condition of hADSCs can be acquired by planting into microwells with different diameters, which can significant affect the rate of apoptosis under this three dimentional cultivate condition. Thinner layer of stack may reduce the rate of apoptosis.