NSCs三维培养及其微流控动态模型的构建
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摘要
神经干细胞(Neural stem cells, NSCs)是一类能够自我更新,并具有多向分化潜能的细胞。它的发现为深入研究神经系统的发育、神经退行性疾病治疗以及受损神经组织的临床移植带来新的希望。而要实现NSCs的各种潜能则必须对其各种生理生化特性有正确的认识与了解。因此,体外构建与体内微环境相似的NSCs模型是深入研究NSCs各种生理机制的重要方法与手段,已成为神经科学与神经组织工程研究领域的热点。
传统的NSCs体外细胞模型主要采用的是二维单层贴壁培养法和“神经球”悬浮培养法,细胞无法真正体现其在体内的生物学特性与功能。而以生物支架为基础的细胞三维培养则被认为更加接近哺乳动物体内真实微环境,已经成为体外研究NSCs增殖与分化机制、药物及环境毒素筛选与评价以及构建神经组织替代物的首选模型。目前,体外构建NSCs三维类神经组织的研究,在国内外还处于实验探索阶段,没有标准化的实验方法。因此,本文主要在NSCs体外三维培养方式及其生长与分化调控方面,开展了以下工作：
(1)获取原代小鼠NSCs(mNSCs)并对其加以鉴定。通过原代培养方法从孕龄14d的昆明种小鼠胎脑的海马组织中获得mNSCs,考察了5个不同的接种密度对mNSCs生长趋势、所形成的“神经球”大小的分布以及扩增倍数的影响。最终确定了原代mNSCs的最佳接种密度为1×105cells/mL。传代培养的mNSCs能够稳定表达NSCs特征蛋白(Nestin和Sox2),经10%胎牛血清诱导能够分化成神经元(β-tubulin-Ⅲ阳性)、星形胶质细胞(GFAP阳性)以及少突胶质细胞(O1阳性),表明本实验分离获得的mNSCs具有自我更新与多向分化潜能。
(2)筛选并确定一种促进nNSCs体外扩增的无血清培养基MA。三维模型需要足够的细胞数量,因此本研究采用正交试验筛选并确定了能够提高mNSCs扩增倍数的培养基添加组分及其浓度与比例,MA显著地促进了mNSCs的增殖,细胞在对数增长期的最大密度可达到(7.25±0.23)×105cells/mL (p<0.01),是初始接种密度((1.0±0.1)×105cells/mL)的7.25倍。同时,免疫荧光染色显示,MA培养的mNSCs其Nestin的阳性表达率为94.2±3.5%,说明优化后的培养基能很好地维持mNSCs的干细胞特性。同时,实验结果显示,MA中葡萄糖/谷氨酰胺配比对mNSCs生长有良好的促进作用,进一步确定了MA是一种适用于mNSCs体外扩增的新型优良培养基。
(3)提出一种基于两步培养的体外构建mNSCs三维静态模型的实验方法。该方法以0.5mg/mL的Ⅰ型胶原水凝胶为支架材料,采用两步培养法模拟体内神经发生的不同阶段的生理微环境。培养初期采用添加了EGF/bFGF/BSA/Lipids的DMEM/F12/RPMI-1640的促mNSCs增殖的培养基MA,待细胞形成直径至50-100μm左右的团簇时,将培养基MA更换为含有bFGF和BDNF生长因子的NB/B27条件培养基(MC)。团簇中的细胞开始沿着支架迁移,直到彼此相连形成神经组织样结构。此构建物内细胞生长状态良好且分布均匀,活率在82%左右,14.17±2.62%细胞仍保持mNSCs特性,而神经元比率为40.93±1.85%,星形胶质细胞比率为27.13±1.63%。
(4)建立了一种基于微流控技术的NSCs三维动态培养方法。mNSCs三维培养技术与微流控技术相结合,设计并建立了一套适合nNSCs三维生长的微柱阵列式微流体芯片培养系统。实现了以胶原水凝胶为支架材料的mNSCs三维复合物在微流体芯片上的构建与生长。结果显示,利用静态培养条件下建立的两步法在芯片上构建的mNSCs三维复合物中有NSCs特征蛋白Nestin的表达,同时具备分化成神经元和星形胶质细胞的能力；对一个培养周期内的细胞代谢产物进行取样分析,芯片三维培养体系中乳酸的浓度始终稳定在1.0mmol/mL以内,谷氨酰胺浓度维持在2.0mmol/mL左右,为mNSCs的生长提供一个稳定的生长环境；而三维静态培养条件下乳酸浓度随着半量换液在2.5～4.5mmol/mL间波动,谷氨酰胺浓度也会随着培养时间不断消耗需要人工补加。这种两步法构建芯片式mNSCs三维模型,操作简便,重复性好,成功率高。便于倒置显微镜下观测,有望成为新型药物筛选或环境毒素监测的神经组织替代物。
Neural stem cells (NSCs) are self-renewable and have the potential to differentiate in multi-directions. The discovery of NSCs has brought great hope for further research on the development of the nervous system, the treatment of neurodegenerative diseases, and clinical transplantations. However, the mechanisms of NSCs proliferation and differentiation are still not well understood. Constructing NSCs model in vitro can be a valuable tool for exploring these mechanisms, which may contribute a lot to the fields of neuroscience and neural tissue engineering.
Traditional in-vitro NSCs culture methods include monolayer culture and the "neurosphere" suspension culture. However, these methods fail to embody the biological characteristics and functions of NSCs in vivo. Growing cells in three-dimensional (3D) matrix is considered a more meaningful method that better simulates the microenvironment of mammalian brain. Constructing3D NSCs model in vitro would provide great value to theoretical and practical applications for promoting the studies of mammalian nervous system development, clinical transplantation of nervous tissues, and drug screening. Currently, the research on3D NSCs culture is still at the stage of experimental research, and there are no standard experimental methods. This paper mainly focuses on three-dimension NSCs culture in microfluidic chip systems, and relevant proliferation and differentiation control issues. For this purpose, the following work has been carried out.
Mouse NSCs (mNSCs) were isolated from the hippocampus of embryonic day-14Kunming mice fetal brain by the method of primary culture. Then, the growth trend and the distribution of neurosphere sizes of five mNSCs suspension cultures with different cell density were investigated. The results showed that the best inoculation density of the primary mNSCs was1×105cells/mL. Subcultured cells could stably express NSCs characterized protein (Nestin and Sox2) and were able to differentiate into neurons (β-tubulin-positive), astrocytes (GFAP-positive). and oligodendrocytes (01-positive) induced by10%fetal calf serum (FCS) medium.
After careful screening, one serum-free medium (MA) was selected for mNSCs amplification in vitro. In order to obtain enough mNSCs for constructing the three-dimension NSCs model, orthogonal tests were used to screen and determine the optimum concentration and ratio of the added components in culture medium MA that can promote mNSCs amplification. The tests displayed that the maximum cell amplification result of the mNSCs cultured in MA can be as high as (7.25±0.23)×105cells/mL (p<0.01).7.25times of the initial inoculum density ((1.0±0.12)×105cells/mL). Besides, immunofluorescence staining showed that the Nestin positive ratio of the mNSCs cultured in the MA was94.2±3.5%. which indicated that the mNSCs cultured in MA can maintain the stem cell characteristics. Also, the glucose/glutamine ratio in MA was proved to be perfect for the growth and metabolism of mNSCs. Which reconfirmed MA to be an excellent medium which was appropriate for mNSCs amplification in vitro.
A two-step method for constructing3D mNSCs model under static conditions in vitro is proposed. The two-steps culture method was used to simulate the physiological environment of different phases of neurogenesis in vivo. The3D scaffold was0.5ing/ml, type1collagen hydrogel based. Firstly, the medium consisting of DMEM/F12/RPMI1640(1:1:1) supplemented with EGF/bFGF/BSA/Lipids was used to expand the mNSCs embedded in collagen in gel in96-well plates until the average diameter of cell clusters reached50-100μm. Secondly, the initial medium was replaced by medium MC. which is NB/B-27supplemented with bFGF and BDNF. The results showed that the cells in collagen presented neural-like morphology and maintained live cell rate around82%. The cell-collagen constructs were examined by immunofluorescence and immunohistochemistry tests, which showed14.17±2.6%cells still maintained the character of mNSCs.40.93±1.8%cells differentiated into neurons, and27.13±1.6%cells differentiated into astrocytes. Our3D neural-like tissue constructs were similar to the neural tissues in morphology and cell compositions.
Finally, the lnicrofluidic cell culture system was used for the three-dimension mNSCs culture. A microfluidie chip with micro-fabricated pillar arrays was designed for the in-situ formation and immobilization of3D mNSCs-collagen construction. The cell-collagen constructs in mierolluidic chip were detected by immunofluorescence test after7days of culture. And. the concentration changes ol lactic acid and glutamine in the3D culture systems were also monitored. The results showed that part of the eells in the center of the3D constructs expressed Nestin protein, and others differentiated into neurons and aslrocytes. which indicated that mNSCs can be self-renewable and differentiate under engineered3D microfluidics culture system. The concentration of lactic acid was steadily controlled below1.0mmol/ml and the concentration ofglutamine was remained around2.0mmol/tnL. which proves that the mierolluidic cell culture system can provide a stable microenvironment for3D mNSCs. In contrast, under the static culture condition, the concentration of lactic acid fluctuated between2.5～4.5rnmol/mL with batch and medium exchange modes, and the concentration of glutamine decreased with cells consumption and needed to be supplemented constantly. The two-step method is easy, stable and fast in building3D mNSCs models on microfluidic chip. And. the3D cell constructs in the chip can be observed clearly under inverted microscope. More importantly, the3D mNSCs model proposed here is similar to the neural tissue in morphology and cell compositions. Therefore, it has great potential for safety pharmacology, drug discovery and toxicity testing.
传统的NSCs体外细胞模型主要采用的是二维单层贴壁培养法和“神经球”悬浮培养法,细胞无法真正体现其在体内的生物学特性与功能。而以生物支架为基础的细胞三维培养则被认为更加接近哺乳动物体内真实微环境,已经成为体外研究NSCs增殖与分化机制、药物及环境毒素筛选与评价以及构建神经组织替代物的首选模型。目前,体外构建NSCs三维类神经组织的研究,在国内外还处于实验探索阶段,没有标准化的实验方法。因此,本文主要在NSCs体外三维培养方式及其生长与分化调控方面,开展了以下工作：
(1)获取原代小鼠NSCs(mNSCs)并对其加以鉴定。通过原代培养方法从孕龄14d的昆明种小鼠胎脑的海马组织中获得mNSCs,考察了5个不同的接种密度对mNSCs生长趋势、所形成的“神经球”大小的分布以及扩增倍数的影响。最终确定了原代mNSCs的最佳接种密度为1×105cells/mL。传代培养的mNSCs能够稳定表达NSCs特征蛋白(Nestin和Sox2),经10%胎牛血清诱导能够分化成神经元(β-tubulin-Ⅲ阳性)、星形胶质细胞(GFAP阳性)以及少突胶质细胞(O1阳性),表明本实验分离获得的mNSCs具有自我更新与多向分化潜能。
(2)筛选并确定一种促进nNSCs体外扩增的无血清培养基MA。三维模型需要足够的细胞数量,因此本研究采用正交试验筛选并确定了能够提高mNSCs扩增倍数的培养基添加组分及其浓度与比例,MA显著地促进了mNSCs的增殖,细胞在对数增长期的最大密度可达到(7.25±0.23)×105cells/mL (p<0.01),是初始接种密度((1.0±0.1)×105cells/mL)的7.25倍。同时,免疫荧光染色显示,MA培养的mNSCs其Nestin的阳性表达率为94.2±3.5%,说明优化后的培养基能很好地维持mNSCs的干细胞特性。同时,实验结果显示,MA中葡萄糖/谷氨酰胺配比对mNSCs生长有良好的促进作用,进一步确定了MA是一种适用于mNSCs体外扩增的新型优良培养基。
(3)提出一种基于两步培养的体外构建mNSCs三维静态模型的实验方法。该方法以0.5mg/mL的Ⅰ型胶原水凝胶为支架材料,采用两步培养法模拟体内神经发生的不同阶段的生理微环境。培养初期采用添加了EGF/bFGF/BSA/Lipids的DMEM/F12/RPMI-1640的促mNSCs增殖的培养基MA,待细胞形成直径至50-100μm左右的团簇时,将培养基MA更换为含有bFGF和BDNF生长因子的NB/B27条件培养基(MC)。团簇中的细胞开始沿着支架迁移,直到彼此相连形成神经组织样结构。此构建物内细胞生长状态良好且分布均匀,活率在82%左右,14.17±2.62%细胞仍保持mNSCs特性,而神经元比率为40.93±1.85%,星形胶质细胞比率为27.13±1.63%。
(4)建立了一种基于微流控技术的NSCs三维动态培养方法。mNSCs三维培养技术与微流控技术相结合,设计并建立了一套适合nNSCs三维生长的微柱阵列式微流体芯片培养系统。实现了以胶原水凝胶为支架材料的mNSCs三维复合物在微流体芯片上的构建与生长。结果显示,利用静态培养条件下建立的两步法在芯片上构建的mNSCs三维复合物中有NSCs特征蛋白Nestin的表达,同时具备分化成神经元和星形胶质细胞的能力；对一个培养周期内的细胞代谢产物进行取样分析,芯片三维培养体系中乳酸的浓度始终稳定在1.0mmol/mL以内,谷氨酰胺浓度维持在2.0mmol/mL左右,为mNSCs的生长提供一个稳定的生长环境；而三维静态培养条件下乳酸浓度随着半量换液在2.5～4.5mmol/mL间波动,谷氨酰胺浓度也会随着培养时间不断消耗需要人工补加。这种两步法构建芯片式mNSCs三维模型,操作简便,重复性好,成功率高。便于倒置显微镜下观测,有望成为新型药物筛选或环境毒素监测的神经组织替代物。
Neural stem cells (NSCs) are self-renewable and have the potential to differentiate in multi-directions. The discovery of NSCs has brought great hope for further research on the development of the nervous system, the treatment of neurodegenerative diseases, and clinical transplantations. However, the mechanisms of NSCs proliferation and differentiation are still not well understood. Constructing NSCs model in vitro can be a valuable tool for exploring these mechanisms, which may contribute a lot to the fields of neuroscience and neural tissue engineering.
Traditional in-vitro NSCs culture methods include monolayer culture and the "neurosphere" suspension culture. However, these methods fail to embody the biological characteristics and functions of NSCs in vivo. Growing cells in three-dimensional (3D) matrix is considered a more meaningful method that better simulates the microenvironment of mammalian brain. Constructing3D NSCs model in vitro would provide great value to theoretical and practical applications for promoting the studies of mammalian nervous system development, clinical transplantation of nervous tissues, and drug screening. Currently, the research on3D NSCs culture is still at the stage of experimental research, and there are no standard experimental methods. This paper mainly focuses on three-dimension NSCs culture in microfluidic chip systems, and relevant proliferation and differentiation control issues. For this purpose, the following work has been carried out.
Mouse NSCs (mNSCs) were isolated from the hippocampus of embryonic day-14Kunming mice fetal brain by the method of primary culture. Then, the growth trend and the distribution of neurosphere sizes of five mNSCs suspension cultures with different cell density were investigated. The results showed that the best inoculation density of the primary mNSCs was1×105cells/mL. Subcultured cells could stably express NSCs characterized protein (Nestin and Sox2) and were able to differentiate into neurons (β-tubulin-positive), astrocytes (GFAP-positive). and oligodendrocytes (01-positive) induced by10%fetal calf serum (FCS) medium.
After careful screening, one serum-free medium (MA) was selected for mNSCs amplification in vitro. In order to obtain enough mNSCs for constructing the three-dimension NSCs model, orthogonal tests were used to screen and determine the optimum concentration and ratio of the added components in culture medium MA that can promote mNSCs amplification. The tests displayed that the maximum cell amplification result of the mNSCs cultured in MA can be as high as (7.25±0.23)×105cells/mL (p<0.01).7.25times of the initial inoculum density ((1.0±0.12)×105cells/mL). Besides, immunofluorescence staining showed that the Nestin positive ratio of the mNSCs cultured in the MA was94.2±3.5%. which indicated that the mNSCs cultured in MA can maintain the stem cell characteristics. Also, the glucose/glutamine ratio in MA was proved to be perfect for the growth and metabolism of mNSCs. Which reconfirmed MA to be an excellent medium which was appropriate for mNSCs amplification in vitro.
A two-step method for constructing3D mNSCs model under static conditions in vitro is proposed. The two-steps culture method was used to simulate the physiological environment of different phases of neurogenesis in vivo. The3D scaffold was0.5ing/ml, type1collagen hydrogel based. Firstly, the medium consisting of DMEM/F12/RPMI1640(1:1:1) supplemented with EGF/bFGF/BSA/Lipids was used to expand the mNSCs embedded in collagen in gel in96-well plates until the average diameter of cell clusters reached50-100μm. Secondly, the initial medium was replaced by medium MC. which is NB/B-27supplemented with bFGF and BDNF. The results showed that the cells in collagen presented neural-like morphology and maintained live cell rate around82%. The cell-collagen constructs were examined by immunofluorescence and immunohistochemistry tests, which showed14.17±2.6%cells still maintained the character of mNSCs.40.93±1.8%cells differentiated into neurons, and27.13±1.6%cells differentiated into astrocytes. Our3D neural-like tissue constructs were similar to the neural tissues in morphology and cell compositions.
Finally, the lnicrofluidic cell culture system was used for the three-dimension mNSCs culture. A microfluidie chip with micro-fabricated pillar arrays was designed for the in-situ formation and immobilization of3D mNSCs-collagen construction. The cell-collagen constructs in mierolluidic chip were detected by immunofluorescence test after7days of culture. And. the concentration changes ol lactic acid and glutamine in the3D culture systems were also monitored. The results showed that part of the eells in the center of the3D constructs expressed Nestin protein, and others differentiated into neurons and aslrocytes. which indicated that mNSCs can be self-renewable and differentiate under engineered3D microfluidics culture system. The concentration of lactic acid was steadily controlled below1.0mmol/ml and the concentration ofglutamine was remained around2.0mmol/tnL. which proves that the mierolluidic cell culture system can provide a stable microenvironment for3D mNSCs. In contrast, under the static culture condition, the concentration of lactic acid fluctuated between2.5～4.5rnmol/mL with batch and medium exchange modes, and the concentration of glutamine decreased with cells consumption and needed to be supplemented constantly. The two-step method is easy, stable and fast in building3D mNSCs models on microfluidic chip. And. the3D cell constructs in the chip can be observed clearly under inverted microscope. More importantly, the3D mNSCs model proposed here is similar to the neural tissue in morphology and cell compositions. Therefore, it has great potential for safety pharmacology, drug discovery and toxicity testing.
引文
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