细胞培养用琼脂微球的制备与初步应用

Preparation and Preliminary Application of Agar Microspheres for Cell Cultures

用4%的琼脂溶液作为水相,环己烷为油相,司盘-80为乳化剂,采用乳化分散法制备了琼脂微球,再通过化学交联法固化.用DEAE-HCl修饰了微球表面的电荷密度,并通过正交试验优化了改性工艺,最终制得细胞培养用琼脂微球.通过对比研究了Cytodex1微载体与琼脂微球的表征及细胞培养效果.结果显示,最优改性工艺为:加入微球2倍体积4.5 mol/L的Na OH溶液,再加入微球2倍体积2.5 mol/L的DEAE-HCl溶液,在搅拌状态下加热至60℃,密闭反应4 h.最终制得琼脂微球表面电荷密度为4.00 mmol/100 g,与Cytodex1微载体的4.05 mmol/100g几乎相当.静置培养时,四种细胞在两种微球上的贴壁率和伸展性各有优劣.悬浮培养BHK-21细胞时,Cytodex1微载体表面出现一定程度的"空球"现象,而琼脂微球表面细胞较均匀地伸展.培养至120 h时,两种微球表面的细胞均达到4.5×107cells.继续培养,Cytodex1表面的细胞数开始下降,而琼脂微球表面仍增殖至168 h时才开始下降.综上所述,自制琼脂微球具备作为细胞培养用材料的条件.

4% agar solution was used as the water phase, a certain volume ratio of cyclohexane as oil phase, span - 80 as the emulsifier, the agar microspheres were prepared by emulsifying dispersion method. Then the chemical crosslinked method was used. At last, orthogonal method was used to optimize the process of Surface charge modification. The contrast research of characterization and cell culture between Cytodexl microcarriers and agar microspheres. Results show that the optimal modification process is： Adding two times volume 4.5 mol/L of NaOH solution, then 2 times volume 2.5 mol/L DEAE HCL solution into microspheres, stirring in the state of heating and 60 ℃, sealed reaction 4 h. Surface charge density of AGAR microsphere was 4.00 tendency for 100 g, was almost of Cytodexl microcarrier （4.05 tendency/100 g）. When cells were stationary culture on the two kinds of microspheres, adherent rate and the extension of four types of cells all had advantage and disadvantage. BHK - 21 cells were in suspension culture, Cytodexl microcarrier surface appeared ＂empty＂ phenomenon, and on AGAR microsphere surface, cells were uniformly. Culturing to 120 h, cells on the surface of the two kinds of microspheres were averaging 4.5 × 107 cells, then cells on the surface of Cytodexlthe cells began to decline, while cells on surface of AGAR microsphere were still proliferation. Summarily, homemade gelatin microspheres to have material for cell culture conditions.