多能干细胞的海藻酸钠封装使用同轴喷嘴

摘要

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

摘要

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N₂ gas. The controllable capsule diameter must be larger than 500 µm because too high a flow rate of N₂ gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl₂ causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

引言

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient¹. However, conventional dish culture is only able to obtain 1 × 10⁵ cells/cm², thus requiring 1 m² of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier² and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells³. Indeed, suspended cells are exposed to shear stress, which causes cell damage³ or differentiation⁴.

Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose⁵, PEG⁶, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg−Ca hydrogel is formed immediately after dropping alginate solution into a CaCl₂ solution and is also readily digested by enzymes or chelating reagents.

Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N₂ gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N₂ and concentration of both CaCl₂ and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

研究方案

1.准备材料

1. 准备10毫米的HEPES缓冲。将pH调节至7.0，在室温，添加NaCl至0.9％。
2. 制备5％藻酸盐溶液和10mM EDTA的溶液通过混合1.1制备的HEPES缓冲盐水。将pH调节至7.0，在室温。
3. 高压釜中加入试剂（1.1，1.2）20分钟，在121℃。
4. 制备层状用1.2明胶包衣60毫米菜- 2.0×10 ⁶的小鼠胚胎成纤维细胞（MEF）细胞，其内含有10％ES-合格FBS和丝裂霉素C的10微克/毫升90当作饲养细胞温育 - 120分钟。
5. 保持小鼠诱导多能干细胞（iPS）在与饲养细胞在5毫升含有的ES-合格的FBS 20％，50μM2-巯基乙醇，非必需氨基酸和抗生素的DMEM高葡萄糖存在下的菜（青霉素G钠，硫酸链霉素100微克/毫升100单位/毫升，及amphoteri的250纳克/毫升霉素B）中。
6. 种子4×10 ⁵个iPS细胞在明胶包被60mm培养皿孵育它们在37℃和5％的CO _2。每天孵化过程中改变培养基。后3 -培养的第4天，trypsinize细胞1分钟用500微升含有0.02％EDTA的0.05％胰蛋白酶在37℃和5％的CO _2。在此之后，通过敲击培养皿十倍分离细胞。
7. 加入2.5的10ml DMEM与ES-合格FBS和抗生素（以下离解小鼠iPS细胞成单个细胞通过移液以1000微升微量三次）离心细胞悬浮液10％，在160×g下3分钟，除去上清液，并计数细胞。以下细胞计数，补种在明胶涂覆的培养皿中收集的细胞，并温育30分钟以从MEF细胞分离iPS细胞。计数细胞后（通常为1 - 3×10 ⁶个细胞/皿可以被收集），上补种4×10 ⁵个iPS细胞这道菜。

2.电池封装成海藻酸钠水凝胶胶囊

1. 通过在1.5和1.6中描述的相同的方法收集细胞。离心后，在160×g下3分钟，三次洗涤的iPS细胞沉淀用HEPES缓冲盐水中。后重新悬浮的细胞在HEPES缓冲盐水，过滤细胞悬浮液通过40微米的细胞过滤以除去大的聚集体，这可能否则堵塞喷嘴。
2. 混合料2 ml的细胞悬浮液中的HEPES缓冲盐水和3ml 5％海藻酸钠-Na溶液。最后5毫升细胞悬浮在3％以内海藻酸钠-Na溶液中获得。细胞密度优选大于10 ⁶个细胞/ ml。
3. 收集的细胞悬浮到5ml的注射器之后，注射器上安装同轴喷嘴（ 图1A，B）和设置它们上的注射泵。 EMIT N ₂气流量（低于1L /分钟）通过同轴喷嘴的外针和通过内喷嘴排出的细胞悬浮液。
4. 收集液滴加到250ml 0.5％的CaCl ₂溶液并等待10 - 20分钟进行凝胶化，在60-90转，同时搅拌。

3.治疗藻酸胶囊（可选）

1. 孵育海藻酸钠钙胶囊在0.05％（重量/体积）聚-l-赖氨酸（PLL）中5分钟，溶液在37℃和5％的CO _2。
2. 洗涤用HEPES缓冲的盐水的胶囊后，孵育他们在DMEM含有10％FBS，以中和在其表面上的电荷。利用它们作为包衣胶囊（ 图1C）。
3. 孵育在含有10mM EDTA的5分钟的HEPES缓冲盐水的胶囊。在EDTA处理的胶囊被用作中空胶囊（ 图1C）。

4.文化和收集细胞的藻酸胶囊

1. 孵育封装细胞以75rpm摇动在37℃和5％CO ₂的10天。
2. 收集和孵化在10mM EDTA的胶囊在37℃和5％CO ₂的10分钟。在1,000×g下3分钟收集来自藻酸胶囊不带PLL处理细胞通过离心。
3. 5000×g下5分钟 - 如果藻酸胶囊与PLL处理，通过移液器上下吹吸10倍左右与连接到一个注射器中，并离心它在1000的针打破海藻酸钠-PLL膜。针直径应比胶囊的尺寸和25克（260微米）的针在本实验中使用的低级（600微米）
4. 离心后，从严从破碎的海藻酸钠-PLL膜，如果你想收集样品的mRNA从细胞中收集细胞沉淀。剩余的海藻酸钠-PLL膜可能阻止mRNA纯化。

结果

在协议2.5，排出的藻酸盐溶液构成的球状立即排出后（ 图2A - H）。若悬浮液排出用的N ₂流动速率大于1L /分下，液滴的尺寸是均匀的（ 图2I）。然而，如果N ₂流量低于1L /分高时，液滴分解（ 图2G，白色带箭头）和液滴的尺寸变得不均匀（ 图2J）。鉴于此，它是难以制备的液滴比使用此方法为500μm以下。

在胶?...

讨论

封装文化可以直接悬浮培养进行比较。悬浮培养是一个更简单的方法，以获得大量的多能干细胞比包封方法。然而，控制在悬浮培养中的细胞的凝集是仍然具有挑战性。在封装方法，蜂窝聚集在胶囊有限的，因此可以很好地控制。先前的出版物表明，包封的细胞形成大小均匀的聚集体，而大细胞团块出现在自由悬浮培养^7。此外，先前的一份报告显示，强烈搅拌导致PS细胞的直接悬浮?...

材料

Name	Company	Catalog Number	Comments
Mouse embryo fibroblast	Cell Biolabs	SNL 76/7
Mouse induced pluripotent stem cell	RIKEN Bio resorce centre	iPS-MEF-Ng-20D-17
DMEM high-glucose	GIBCO	11995
ES qualified  FBS	GIBCO	16141079
Antibacterial Antibiotics	GIBCO	15240
Nonessential Amino Acid	GIBCO	11140
2-mercaptoethanol	GIBCO	21985-023
ESGRO Leukemia Inhibitory Factor	Merck Millipore	ESG1107
Trypsin/EDTA	GIBCO	25300
26 G/16 G needle	Hoshiseido
10 ml Syringe	TERUMO	SS-10ESZ
Sodium  Chloride	Wako	191-01665
HEPES	SIGMA	H4034
Sodium Alginate	Wako	194-09955
Calcium Chloride	Wako	039-00475
Poly-L-lysine (MW = 15,000 - 30,000)	SIGMA	P7890
EDTA	DOJINDO	345-01865
Sylinge pump	AS ONE
Microscope	Olympus	IX71
Microscope	Leica	DM IRB
Hispeed camera	nac image technology	Memrecam HX-3