薯条和小费

Ayako Yamada^{一百二十三},Jean-Louis Viovy^{一百二十三},Catherine Villard^{一百二十三}和圣_潘尼除垢剂^{一百二十三}

^一居里物理实验室，Institut CuriePSL Research University,CNRS UMR168,Paris,France.

^二Sorbonne Universités,UMPC Univ.巴黎06，Paris,法国

³皮埃尔·吉勒斯·德根尼斯研究所，Paris,法国

Email: ayako.yamada@curie.fr

Why is this useful?

玻璃是一种多用途的化学处理表面，它仍然是迄今为止最常用的表面工程基板（例如micropatterning,surface 新利手机客户端chemistry) or plasma-bonding of PDMS microfluidic devices.在这种基质上进行细胞培养，玻璃底培养皿是为了保持细胞外明确的培养基体积，保护细胞免受污染和介质蒸发。Moreover,它们在光学上比通常用于细胞培养的聚苯乙烯培养皿更适合显微镜观察。Although glass-bottom culture dishes are commercially available (e.g.来自WPI的荧光皿）the presence of plastic walls limits the treatments that can be performed onto the glass bottom surface and those are more expensive ( 5 € per dish;φ 50 mm) than polystyrene dishes (e.g.φ 40 mm dish from TPP,0.5 € per dish).在这个提示中，we describe an easier way than a previous Tip^一把聚苯乙烯培养皿变成玻璃底培养皿，同时保留了在玻璃装配成盘子之前对其进行任何处理的可能性。Note that in this method,the body of the culture dish will be upside down and the lid is thus no longer lifted above the dish opening by lid stoppers.然而，the gas exchange through the gap between the body and its lid seems to be enough to culture cells healthily in this dish.In summary,this Tip provides a low-cost and rapid solution for cell culture in a microfluidic device or on an engineered surface directly in a culture dish,suited for a long-term live cell imaging.

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What do I need?

• φ 40 mm polystyrene tissue culture dish (e.g.TPP #93040)
• φ40 mm盖板滑块（例如Thermo Fisher Scientific #11757065,每张幻灯片～0.2欧元）
• Large screw driver (or a similar tool)
• PDMS基和固化剂的未固化混合物（10:1 w/w）
• 烤箱或热板
• Ferromagnetic metal plate (e.g.PDMS容器盖，optional)
• Cylindrical magnets (optional)

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What do I do?

1. Place a polystyrene culture dish upside down on a surface and hit a few times the center of the dish bottom with the grip of a large screw driver (Fig.1a) until the dish bottom falls apart from the dish wall (Fig.1b）。成功率在9%到10%之间时，底部应该容易下降。Avoid breaking the dish wall by hitting the bottom too strongly.
2. Spread uncured PDMS mixture on a flat substrate (e.g.一个较大的塑料培养皿），并在培养皿边缘（破碎部分）涂上PDMS（图1c).
3. Place the dish (broken part up) on a cover glass slide (Fig.1d) and cure PDMS in an oven or on a hotplate e.g.在80°C下保持10分钟（图1e).
4. 表面处理（例如micro-contact printing) or plasma-bonding of a PDMS chip to the glass surface can be performed after or before the dish assembly (Fig.1F）。

5. 为了保持片上细胞培养的湿度，这道菜可以用例如phosphate buffered saline (Fig.2a).装有芯片或微电池的盘子可以放在一氧化碳中。_二有或无进一步保护的孵化器（图2b).
6. Long-term live cell imaging can be performed using a stage top incubator (Fig.2C）。

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我还应该知道什么？

7. 根据显微镜的支撑类型，it might be necessary to well align the contours of the dish and the glass slide.这可以使用圆柱形磁铁（每盘3个）和铁磁金属板（图3a）在烤箱或加热板上进行PDMS固化时（图3b）。

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确认

这项工作得到法国国家研究机构（ANR）的支持，作为“Avenir投资计划”（参考号：ANR 10-nano 0207）和ERC高级赠款大提琴（FP7-IDEAS-ERC-321107）的一部分。

参考文献

[1] Caballero D,Samitier J为活体细胞成像研究制造细胞培养室的不同策略。Chips and Tips,2014年12月2日（//www.xcmww.com/chipsandtips/2014/12/02/different-strategies-for-the-fabring-of-cell-culture-chambers-for-live-cell-imaging-studies/）

Gabriele Pitingolo^一and Valerie Taly^一

^一INSERM UMRS1147,CNRS SNC 5014,巴黎笛卡尔大学Equipe labellisée Ligue Nationale contre le cancer 2016.Paris,France.

email:gabriele.pitingolo@parisdescartes.fr

Why is this useful?

近年来，几种明胶类型（即GelMA,A or B) have been used in pharmaceutical formulation and tissue engineering due to their excellent biocompatibility,propensity to cell differentiation and availability at low cost.^一The use of gelatin as biomaterial is also advantageous for the possibility to tune the mechanical properties of the substrate,changing the concentration in water and the degree of cross-linking.然而，Transwell^®is the most used permeable support with microporous membranes and is a standard method for culturing cells.^二This commercial type of support has been widely used to study the molecular secretion by different cell types and also to reproduce severalin vitrophysiological barriers (i.e.血脑屏障）。³Transwell^®细胞培养插入很方便，because they are sterile and easy-to-use,but they are very expensive (~ 300 $ for a 12 pack) and possess a limited range of biomaterial properties because they are made from polyester or polycarbonate.此外，as shown by Falanga and colleagues,这些多孔膜通常集成在微流控芯片上进行渗透性研究。⁴

Recently,Yong X.陈等人。proposed an alternative method to prepare a suspended hydrogel membrane platform for cell culture,设计了一个复杂的合成凝胶的协议，并用商用打印机制作了一个由聚乳酸（PLA）聚合物制成的开放式网格结构。^五Our tip shows a novel low-cost method for preparing a cross-liked gelatin membrane as a permeable support useful for potential biological applications.In addition,提议的方案不需要使用复杂的制造技术或昂贵的材料。它使用来自猪皮肤的明胶和甲醛蒸气技术交联明胶膜。作为概念证明，we integrate the gelatin membrane into a microfluidic chip,to show the possibility to develop a platform for comparable studies in static and dynamic conditions.

此外，为了证明制备的明胶膜对培养温度（约37°C）的抗性，我们在孵育前后（2天）测试了力学性能（杨氏模量）。Finally,我们观察了机械性能和结构完整性的保存，使膜可用于细胞培养的研究。

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What do I need?

• Transwell插件
• Porcine gelatin type A
• 甲醛溶液
• 手术刀
• PMMA研磨室或类似

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What do I do?

1. 从Transwell中取出多孔膜^®insert (Fig.1a-1c) or alternatively use a similar homemade support.为了方便这一步是方便使用解剖刀沿整个直径切开膜。

2. 膜移除后，Transwell®支持随时可用。Position the structure at the center of the PMMA chamber (depth 2 mm at least) (Fig 2a) and pour liquid 10% w/v gelatin,without bubbles,onto the PMMA chamber and inside the Transwell^®support (Fig 2b).稳定2分钟后，put the system in the fridge,for at least 10 minutes.

3. After the gelation time (10 minutes at 4°C) it is possible to remove the formed gelatin membrane from the PMMA chamber,借助手术刀促进分离（图3a-3b）。如图3c所示，明胶膜看起来非常平坦，an ideal characteristic for cell cultivation (Fig 3c).To guarantee the preservation of the mechanical properties during the cell culture step,cross-link the gelatin membrane using the classical protocol "cells-biocompatible",such as glyceraldehyde⁶,甲醛^七and glutaraldehyde⁸methods or natural products such as genipin.^九

4. In this case,采用气相甲醛法将制备的明胶膜交联，得到水溶性较低的体系。higher mechanical strength and stability against enzymatic degradation.我们将明胶膜暴露在甲醛蒸气中1天。在图4a中，我们显示了差异，培养48小时后，between a sample cross-linked (left) and not (right).交联明胶膜的最终深度约为1 mm，然而，it is possible to change the depth tuning the liquid gelatin amount.Finally,我们计算，before and after the incubation time,交联明胶膜的杨氏模量，observing a similar value of 40 kPa (compression test by using hydraulic testing system Instron DX).

5. Integration of the gelatin membrane into a microchip.In this section,we detail the integration of the gelatin membrane into a microfluidic chip.As example,we used the same geometry proposed in our previous work⁴,to make a device for a permeability experiment (Figure 5a).如图5b所示，just pour the liquid gelatin into the smaller drilled microchannel,using a pipette to form a thin uniform layer of gelatin (Figure 5c).After gelation at 4° C,cross-link the formed gelatin membrane using the previously described method,结果如图5d所示。为了连接不同的PMMA-PDMS基板，我们在这里提出了一种我们小组最近开发的磁性方法。¹⁰,通过物理化学应力保护明胶膜，如溶剂蒸发和等离子粘合。Figure 5e shows the final chip.

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结论：In this tip,a novel biocompatible gelatin permeable support was obtained by using a simple and low cost fabrication method.可采用气相甲醛法或其他化学交联方法对整体明胶膜进行交联，作为细胞培养的潜在支架。此外，通过改变明胶的初始浓度，可以调节渗透膜的杨氏模量和厚度。the degree of cross linking and the amount of liquid gelatin.Finally,we showed the integration of the gelatin membrane into a modular microchip.因此，we propose an easy and low cost method to prepare a permeable gelatin membrane for cell biology and for other applications.

确认

This work was carried out with the support of the Pierre-Gilles de Gennes Institute equipment ("Investissements d'Avenir" program,reference: ANR 10-NANO 0207).

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