人垂体腺瘤细胞的三维海藻酸钠珠文化
Summary
Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.
Abstract
三维培养方法进行说明，其中主垂体腺瘤细胞生长在藻酸盐珠。海藻酸钠是从棕色海藻衍生的聚合物。简言之，将肿瘤组织切成小块并用胶原酶和胰蛋白酶提交酶促消化。接着，获得的细胞悬浮液。肿瘤细胞悬浮液与1.2％的藻酸钠混合，并落入氯化钙溶液，和藻酸盐/细胞悬浮液凝胶化上与氯化钙 2接触，以形成球形珠。嵌在藻酸盐珠将细胞用由有20％FBS富集培养基中提供营养物供给。在藻酸盐珠的三维培养保持腺瘤细胞的活力为长时间，最多四个月。此外，细胞可以从藻酸盐通过洗涤柠檬酸钠珠粒中释放并接种于玻璃盖玻片上用于进一步免疫细胞化学分析。使用CEL的升培养模式可以使用最少的解体细胞骨架的固定和可视化。总之，藻酸盐珠用于维修垂体腺瘤细胞的可靠培养系统。
Introduction
三维支架已在细胞培养被广泛使用，因为它们提供用于培养细胞的三维结构支撑和细胞-细胞通讯1的维护。天然衍生的聚合物材料已用于制造三维支架，包括I型胶原蛋白，壳聚糖和藻酸盐。海藻酸钠是从褐色海藻Macrocystispyrifera（海带）的天然聚合物。该聚合物是由β-D-甘露糖醛酸（M）和α-L-古洛糖醛酸（G）的2和形式在某些二价阳离子，如钙和钡的存在下是稳定的凝胶的重复单元。氯化钙（ 氯化钙 ）溶液通常用作交联剂以形成藻酸盐珠。海藻酸钠溶液滴入氯化钙立即形成三维球凝胶。当藻酸盐溶液与细胞混合，将细胞封装在藻酸盐bEADS 3。
藻酸盐具有已启用它的属性被用作用于多种细胞，包括软骨4，骨骼肌成肌细胞5和神经干细胞6的包封的基质。它是一种惰性材料，并允许营养物，氧气和维持细胞的存活和功能的代谢产物的扩散;此外，不同于其他基于凝胶的培养系统，藻酸盐珠内培养的细胞，也可以从该支架使用钙螯合剂，如柠檬酸钠解放，然后将细胞可以收获进一步调查7。
垂体腺瘤通常是良性肿瘤低增殖率。大鼠垂体腺瘤细胞系已经在二维系统 8成功培养。然而，这种文化分泌人垂体细胞瘤是不是一个高效的开发;分散垂体肿瘤细胞生长不佳在培养中，细胞呈现出有限的附着和铺展，并将细胞通常形成在培养皿9,10-漂浮聚集。
已进行了多种尝试，以获得肿瘤垂体细胞悬浮液，包括酶和机械分散方法11，一个单独机械分散方法12和肿瘤外植体10的培养。用这些方法中，不同的作者获得可行的粘附培养不同的时间段。肿瘤分泌细胞的能力在这些两维系统生存取决于肿瘤的类型和它们的增殖率9。但是，在长期培养，与成纤维细胞表型细胞为主11,13。本文介绍了从封装在藻酸盐珠垂体腺瘤细胞进一步释放他们的海藻酸钠支架，能够进行详细的分析获得原代培养的方法它们的细胞生物学，例如，其细胞骨架的安排的各个方面。
Protocol
Representative Results
Discussion
该协议有几个关键步骤。第一是珠粒度均匀性，这是需要保持营养物和气体的扩散的相同的条件下在所有的细胞培养物。根据我们的经验，采用了21g的针头，使藻酸盐可用于收购珠均匀大小的有效文化。第二个重要因素是从针的距离;这个距离必须不超过从钙溶液5cm至避免变形珍珠和死细胞由于与钙溶液的表面上的藻酸盐/细胞溶液流的碰撞。第三关键步骤是在玻璃搅拌在其中珠被丢弃，以防止它?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Mr. O. Rios for his technical assistance.
Materials
| Reagent | |||
| 199 culture medium | Invitrogen | 31100-027 | For culture, warm in a 37 °C water bath before use |
| Fetal bovine serum | PAA | A15-751 | |
| Sodium bicarbonate (NaHCO3) | Merck | 106329 | |
| N-(2-Hydroxyethyl) piperazine-N´-2ethanesulfonic acid (HEPES) | Sigma | H-4034 | |
| Penicillin/Streptomycin | PAA | P11-010 | |
| PBS (Dulbecco's Phosphate Buffered Saline) | Invitrogen | 21600-044 | |
| Collagenase type I | Worthington | 4176 | |
| Trypsin | Invitrogen | 27250-018 | |
| Ethylenediaminetetraacetic acid (EDTA) | Research Organics | 3002E | |
| Sorbean trypsin inhibitor | Invitrogen | 17075-029 | |
| DNAse | Worthington | 2139 | |
| Alginic acid, From Macrocystis Pyrifera (Kelp) | Sigma | A-2158 | |
| Calcium chloride (CaCl2) | J.T. Baker | 1332 | |
| Sodium citrate (Na3C6H5O7) | J.T. Baker | 3646 | |
| Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) | Research Organics | 9624P | |
| Polyethylene Glycol 6000 (PEG 6000) | Calbiochem | 528877 | |
| Ethylene glycol tetraacetic acid (EGTA) | Research Organics | 9574E | |
| Magnesium Sulfate (MgSO4) | J.T. Baker | 2500 | |
| Triton-X 100 | Sigma | X100 | |
| Formaldehyde | J.T. Baker | 2106 | |
| Paraformaldehyde | Sigma | P6148 | |
| Ammonium chloride (NH4Cl) | J.T. Baker | 660 | |
| BSA Bovine Serum Albumin IgG-Free | Jackson Immuno Research | 001-000-161 | |
| Phalloidin–Tetramethylrhodamine B isothiocyanate | Sigma | P1951 | Toxic. Use gloves to handle this reagent |
| 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) | Invitrogen | D1306 | Toxic. Use gloves to handle this reagent |
| Sulfuric acid (H2SO4) | J.T. Baker | 9681 | Highly corrosive. Use gloves to handle this reagent |
| Sodium hydroxide (NAOH) | Merck | 1.06498 | Can cause eye and skin irritation.Use gloves to handle this reagent |
| (3-Aminopropyl)triethoxysilane (APTS) | Sigma | A-3648 | |
| Poly-D-lysine hydrobromide | Sigma | P7280 | |
| Trypan blue solution | Sigma | T8154 | |
| Name | Company | Catalog Number | Comments |
| Equipment | Toxic: May cause cancer. Use gloves to handle this reagent | ||
| Rotator | Boekel Scientific | Model 230300 | |
| Centrifuge | DuPont Corporation | Model sorvall TC6 | |
| shaker | Lab-line Instruments | Model 314-820 |
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