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查看更多产品信息 AlgiMatrix™ 96-Well, 3D Culture System, Flat-Bottom Microplate - FAQs (12684015, 12684031)
44 个常见问题解答
AlgiMatrix 基质在不开封的条件下可稳定放置12个月。一旦开封后，整板都需及时用掉。
Gibco Geltrex 基质是含有细胞粘附所需的胶原IV，层粘连蛋白及其他基质蛋白和生长因子的基底膜抽提物。AlgiMatrix 基质只含有不具细胞粘附促进能力的藻酸盐。藻酸盐海绵体中没有能与细胞相互作用的成份。
除了Invitrogen alamarBlue 细胞活力检测试剂（Cell Viability Reagent）之外，AlgiMatrix 基质还能够与Invitrogen PrestoBlue 细胞活力检测试剂，Promega Cell Titer-Glo 试剂或Invitrogen CyQuant Direct Cell Proliferation Assay试剂盒相兼容。具体实验方案与2D细胞培养系统几乎相同，但需要加入纯AlgiMatrix 基质作为空白对照组。
固化缓冲液通过向海绵体系中加入钙成份来发挥作用——加入后使海绵体变硬。解离缓冲液通过去除海绵体系中的钙成份来发挥作用——加入后使海绵体变软。
使用AlgiMatrix 基质的3D细胞培养体系为细胞的生长和行为提供了一个更接近生理的条件，但由于该系统更为复杂，通常也的确会带来更高的结果变异度。您可能需要在实验中设置更多的重复组和对照组。
某些原代细胞（干细胞除外）需要贴壁才能增殖。由于细胞不会粘附于AlgiMatrix 基质，因此尽管这些细胞能够继续培养，但不会增殖而更倾向于分化。我们推荐您以高细胞密度接种原代细胞。
海绵体在干燥时的高度为3-4mm左右。当海绵体重新水化时，其体积本质上是不变的——不会发生膨胀或体积增加的情况，事实上还会由于恢复了水凝胶状态而体积轻微缩小，这一情况在对已接种细胞的海绵体进行短暂离心时尤其明显（按照建议的那样是为了让细胞更多地进入海绵体内部，并让海绵体均匀地分布于培养孔底部）。
用于制备AlgiMatrix 基质的藻酸盐，具有非常亲水的表面，含脂质的细胞膜甚至分子都不会粘附其上。正常细胞在AlgiMatrix 基质中会聚集形成细胞球并分化成结构，但扩增会受到限制，因为大多数正常细胞数目的增加需要以粘附为前提。癌细胞能够在不贴壁的情况下保持扩增，在AlgiMatrix 基质中也是如此（一个例外是胚胎干细胞，这一类型的细胞在AlgiMatrix 基质中会扩增为拟胚体）。
不过，软琼脂与AlgiMatrix基质之间的一个主要区别在于：软琼脂是一种水凝胶，而AlgiMatrix 基质则是拥有较大孔径的海绵体——细胞在AlgiMatrix 基质中比在水凝胶（软琼脂）包裹下能更容易和更迅速地进行生长。从正常细胞中区分癌细胞的经典方法是观察细胞的增殖；癌细胞（如HepG2肝癌细胞）会形成不断扩增的细胞球，而正常细胞所形成细胞球的大小尺寸和数量都不会增加。
最近推出的高纯度藻酸盐产品不会引起之前低纯度产品所面临的敏感性反应。藻酸盐多年来一直在用于包裹例如胰腺B细胞一类的物质以用于体内移植（糖尿病相关研究）而不会引起排异反应。
我们的供应商多年来一直生产药用级海藻酸和透明质酸。虽然这些都是天然的产品，但通过指定获取地点(以海藻酸盐为例)、水温、海藻中要获取哪些部分等其它方面，使用严格的净化操作规程和严格的QC标准，就可以得到非常标准的产品。
侵润研究是对细胞穿透基质能力进行检测的研究，这一检测其模拟了细胞在体内迁移的活性。AlgiMatrix 基质未经修饰的结构中仅含有藻酸盐（源自褐海藻海带），而不含任何体内类似分子物质，所以AlgiMatrix 基质与细胞侵润研究可能不是很相关。
标准的免疫荧光检测能用于3D结构。您即可对AlgiMatrix 基质中的完整细胞球进行直接染色，也可对使用Versene或/55 mM 柠檬酸三钠二水合物溶液溶解掉AlgiMatrix 基质后的细胞球本身进行染色。您也可使用TrypLE 试剂将细胞球进一步解离为单个细胞，再对分散的细胞进行染色。
对于脐带干细胞而言，您可能希望尝试相对较高的接种密度——每个海绵体10万-30万个细胞，并每天或每两天当培养基变“黄”时进行培养基补加。
这种支架非常适合形成三维球体，允许最大限度的细胞间相互作用。根据细胞类型的不同，已建立的细胞系可以通过增加细胞球的大小而扩大到100个细胞左右。以三维形式培养1-2周后，原代细胞和已建立细胞系都将进行分化。
请参见下列专门用于讨论干细胞的参考文献：
Methods Enzymol 420:303 (2006).
Biotechnol Bioeng 88:313 (2004).
使用藻酸盐实际上不会造成任何背景着色。能避免细胞粘附的同一表面性质也使荧光素标记抗体等分子很难粘附，因此实际上未发现任何背景染色效果。
我们内部未对此应用进行测试，通常情况下人们相信如果藻酸盐支架能够在无饲滋养层细胞的条件下支持人体ES细胞的增殖，则应该也能支持小鼠ES细胞的增殖。小鼠与人体ES细胞均会形成拟类胚体，从增殖和分化的角度而言，人们发现在带有较大孔隙的藻酸盐支架中接种对于培养这些结构非常有帮助。
可以，您可使用钙结合液，Versene（乙二胺四乙酸）或柠檬酸钠来溶解基质。剩余的细胞球可通过TrypLE 试剂解离下来，计数，并在其他培养器皿瓶，或新的AlgiMatrix 基质海绵体中进行扩增。随试剂盒附赠的说明手册中对这些操作步骤进行了图解说明。
一旦接种完成，细胞球大约在5至7天内形成。接种过程中会观察到气泡。随着细胞对氧气的逐步消耗，这些气泡将在数天内消失。
轻柔地以一定角度加入培养基。补加细胞时请小心操作，不要搅动基质。
我们展示了以下数据：
聚焦在培养细胞时，可显示细胞已接种的证据。
石蜡包埋和切片结果显示细胞接种到了整个海绵体中。
Gibco Geltrex 基质与胶原（大鼠尾与牛）可作为包被溶液或3D凝胶基质来使用。CELLstart 底物作为一种不含异外源性物质的包被基质，专为ESC的相关应用而研发（作为动物源性的Gibco Geltrex 基质或动物源性的Matrigel 基质的一种替代品底物）。AlgiMatrix 基质是为一种3D支架类的基质，它不支持并不为细胞提供粘附的基础，但而是为细胞球生长提供一个良好的生长环境，后续应用中也可方便易于细胞球的收取操作用于后续应用。
AlgiMatrix Matrix is stable for 12 months unopened. Once opened, the entire plate needs to be used.
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Geltrex Matrix is a basement membrane extract that contains collagen IV, laminin, and other matrix proteins and growth factors that are required for cell adhesion. AlgiMatrix Matrix contains only alginate which does not facilitate cell adhesion. There is nothing in the alginate sponges for the cells to interact with.
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Other than the Invitrogen alamarBlue Cell Viability Reagent, AlgiMatrix Matrix is also compatible with Invitrogen PrestoBlue Cell Viability Reagent and Invitrogen CyQuant Direct Cell Proliferation Assay. The protocol is almost the same as the 2D cell culture, but it needs the blank AlgiMatrix Matrix as a blank control.
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The firming buffer works by adding calcium to the sponge, making it stiffer. The dissolving buffer works by removing calcium from the sponge, making it softer.
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3D cell culture with AlgiMatrix Matrix approaches a more physiological state in terms of cell growth and behavior, but along with that comes the higher variability typically associated with more complex systems. You may need to do more replicates or more controls.
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Some primary cells (with the exception of stem cells) need to adhere in order to expand. As cells won't adhere to AlgiMatrix Matrix, these cells can still be cultured, but will tend to differentiate rather than expand. We recommend that you plate at a high cell density for primary cells.
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The height of the sponge is ~3-4 mm dry. When the sponge is hydrated, its volume is essentially the same - it doesn't swell and increase in size, in fact it becomes slightly bit smaller as it essentially goes back into a hydrogel consistency, especially if the inoculated sponges are centrifuged briefly (as suggested to force cells more into the interior and also to spread the sponge uniformly over the bottom of the well).
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Alginate, from which AlgiMatrix Matrix is made, presents a very hydrophilic surface, and lipid-containing cell membranes and even molecules do not adhere. Normal cells will aggregate to form spheroids and differentiate into structures in AlgiMatrix Matrix, but expansion will be limited since most normal cells need to adhere to increase in number. Cancer cells are able to expand without attachment and do so in AlgiMatrix Matrix (one exception to this is embryonic stem cells, which can expand as embryoid bodies in AlgiMatrix Matrix).
However, here is one major difference between soft-agar and AlgiMatrix Matrix: soft-agar is a hydrogel and AlgiMatrix Matrix is a sponge with relatively large pore sizes, and cells that do grow will be able to do so easier and faster in AlgiMatrix Matrix than when encased in a hydrogel (soft-agar). So a traditional way to tell cancer cells from normal cells is to observe the culture for expansion; cancer cells (example HepG2 hepatocarcinoma) will form expanding spheroids while normal cells will form spheroids that do not increase in size or number.
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The recent more highly purified forms of alginate do not generate a sensitivity reaction as is the case with less pure versions. Alginate has been used for some years to encapsulate, for instance, B-cells of the pancreas for in vivo transplantation (diabetes research) and it does not get rejected.
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Our supplier has been producing pharmaceutical-grade alginate and hyaluroinic acid for years. Although these are natural products, by specifying among other things (in the case of alginate) harvest location, water temperature, which part of the seaweed to harvest, use of stringent purification SOPs, and rigorous QC criteria, a very standard product can be obtained.
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Invasion studies measure the ability of cells to penetrate through a matrix, simulating a metastatic cells activity in vivo. AlgiMatrix Matrix contains only alginate (brown seaweed) with no in vivo-like molecules in the undecorated structure, so invasion may not be a very relevant use for AlgiMatrix Matrix.
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Standard immunofluorescence testing can be performed on 3D structures. The staining can be done with either intact spheroids within the AlgiMatrix Matrix or you can dissolve the AlgiMatrix Matrix using Versene or 55 mM tri-sodium citrate dihydrate and stain the spheroid alone. You can further dissociate the spheroid into individual cells using TrypLE reagent and stain as dissociated cells.
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For umbilical cord stem cells, you might want to try a relatively high inoculation density of 100,000-300,000 cells per sponge with daily or every-other-day re-feeding of cultures, as the medium turns “yellow”.
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This scaffold is great for forming 3D spheroids that allow maximum cell to cell interaction. Established cell lines expand by increasing size of spheroids up to ~100 cells or so depending upon the cell type. Both primary and established cells will differentiate in a 3-dimensional format over 1-2 weeks of culture.
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Please see the following references specifically for stem cells:
- Methods Enzymol 420:303 (2006)
- Biotechnol Bioeng 88:313 (2004)
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There is virtually no background staining with alginate. The same surface characteristic that prevents cells from adhering also makes it very difficult for molecules such as fluoresceinated antibodies to adhere, thus essentially no background staining is observed.
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While we have not tested this in-house, the common belief is that if the alginate scaffolds support proliferation of human ES without feeder cells, they should also support mouse ES. Both mouse and human form embryoid bodies, and seeding within the macro-porous alginate scaffolds have been found very advantageous for these structures, in terms of proliferation and differentiation.
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Yes, start by dissolving the matrix using calcium binding solution, Versene, or sodium citrate. The remaining spheroids can be dissociated with TrypLE reagent, counted, and expanded into other culture vessels or into new AlgiMatrix Matrix sponges. These steps are illustrated in the instruction manual that is supplied with each kit.
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Spheroid formation requires about 5 to 7 days once inoculated. Air bubbles will be observed at the time of inoculation. This will dissipate within several days by the cells consuming the oxygen.
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Add medium gently at an angle. Be careful not to impel the matrix when re-feeding.
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We have shown the following data:
- Focusing on cells in culture shows evidence of inoculation.
- Paraffin embedding and sectioning shows that cells are inoculated throughout the sponge.
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Gibco Geltrex Matrix and collagen (rat tail and bovine) can be used as either a coating solution or a 3D gel matrix. CELLstart Substrate was developed to be used as a xeno-free coating matrix for only ESC applications (as a substitute for Gibco Geltrex Matrix or Matrigel Matrix, which are of animal origin). AlgiMatrix Matrix is a 3D scaffold-type matrix that does not support cell attachment but does provide a good environment for growing spheroids that can be easily harvested for downstream applications.
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