PDMS微流体装置的廉价制造的非等离子键合

Biology
 

Summary

在这段视频中,我们演示了如何使用没有等离子粘接的神经元微流体装置。

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Harris, J., Lee, H., Vahidi, B., Tu, C., Cribbs, D., Cotman, C., Jeon, N. L. Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices. J. Vis. Exp. (9), e410, doi:10.3791/410 (2007).

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Abstract

在这个视频中,我们展示了如何在不使用等离子粘接的神经元微流体装置。在某些情况下,它可能是可取的可逆债券设备,康宁公司的1号玻璃盖。这可能是因为,也许,等离子清洁,不可用的。在其他情况下,它可能需要从玻璃中删除设备后显微镜或为某些类型的免疫的神经元的培养,虽然这是没有必要因为免疫染色的神经元可在设备中删除设备。然而,一些研究人员仍然倾向于删除该设备。在这种情况下,设备可逆的粘接到玻璃盖可能。非等离子设备,而不是作为一个密封紧形成的粘接,也有一些缺点。轴突在某些情况下可能会下的沟槽,而不是通过他们的成长。此外,因为玻璃和PDMS是疏水,液体不容易进入设备使得有必要在次强行进入设备的媒体和其他试剂。通过毛细作用,液体将进入设备,但它需要显着较长相比已血浆保税设备。等离子体清洁玻璃和设备上创建的临时的亲水性收费,以便通过设备后粘接在几秒钟内液体流动。对于非等离子体约束装置,通过设备的液体流动需要几分钟时间。同样重要的是要注意设备与非血浆用于粘接需要先进行消毒,而等离子体处理设备不需要进行消毒,使用前因为等离子体清洁消毒。

Protocol

准备神经元细胞培养的微流体装置

1)清洁康宁第一24毫米x 40毫米玻片

  • 30分钟超声水浴中sonicator玻片
  • 用70%乙醇冲洗玻片
  • 洗净幻灯片DH 2 O 3倍
  • 组织培养罩在几个小时,最好是在一夜之间干幻灯片

注:我们有特殊的金属托盘,我们加载的幻灯片。幻灯片分开单独插槽放置在这个金属架。然后我们就可以放置在一个玻璃皿这些金属装入纸盘,我们与水,在水浴sonicator中进行填写,并超声30分钟。随后的清洗都进行了这盘。

2)准备的PDMS设备

  • 从硅片切割的PDMS模具,打孔PDSM投季度,
  • 清洁的PDMS设备先吹惰性气体(氩气或氮气)。然后用3M苏格兰品牌471磁带升空任何剩余的碎片

注:重要的是要保持设备面对。最初,当我们从硅片切PDMS的距离,与晶圆接触的一面是干净的一面:它包含了微流体通道。我们尝试要小心,因为我们可以以不损坏设备,并保持清洁面朝直到我们准备等离子粘接。

  • 在塑料容器的高压灭菌设备
  • 经过高压灭菌,清洁1号康宁玻璃幻灯片和等离子治疗设备使用一个Harrick品牌等离子清洁, www.harrickplasma.com
  • 放置设备的清洁载玻片上的一面朝下。

该设备是目前保税载玻片等离子。

3)涂层设备与聚- L -赖氨酸(PLL)

  • PLL被添加到每个设备端,并允许流入未来以及通过良好

=> 150μL较小井大井和30μ升PLL PLL的 。它不准确,只要井满。

注意:如果你在一个设备,你会看到4口井,每两个连接。你想,使其流入未来以及通过设备把在一个PLL。

  • 允许流经设备的PLL约10分钟,然后加入更多的PLL的水井,以填补他们
  • 放置至少4小时在孵化器的装置,含有锁相环(隔夜可取)在37 ° C
  • 真空治疗后多余的PLL,但小心,不要吸的设备所有的液体

注意:您不希望引入气泡渠道或分庭。只要真空井多余的PLL。

  • 添加蒸压生署2 O以及每个设备两侧,并允许流量通过毛细作用等以及

图1是一个微流体装置图。请注意蓝(SOMA方)如何连接红(轴突方)连接。当我们说的PLL,或水或媒体,以及每个设备端,我们的意思,例如,是:将150蒸压DH 2 O的 UL在一个蓝色的那么到位150μL灭菌的dh 2 0成一个红井。 ,水(或PLL,或媒体,或细胞),将流经该设备其他相应的井。

图1

图1

注意:请注意,从现在起,当我说“的地方媒体,细胞,水或在TOP井PLL的”,我指的是以上的SOMA将在左边的图和轴突将增长到权利。

  • 吸过的水(再小心不要完全从设备中取出所有的液体),然后加入150μL另一个蒸压DH 2 O接好每一个,并允许它流动到相应的。
  • 将设备包含在一个孵化器2 O卫生署在37 ° C为1小时,然后重复清洗步骤。洗净设备与DH 2 O三次,每次一小时,这是总的。

注意:由于从PLL硼酸可以吸收到的PDMS,在全度妍实验室的一些建议后,我夫妇的孵化设备蒸压生 2过夜的可能性nitial快速冲洗。这将确保所有免费硼酸,将滤出的PDMS前加载的细胞。如果不这样做,有可能到媒体发布的硼酸,随着时间的推移,这可能导致细胞毒性。

  • 添加必要的因素(glutamax,B27,PenStrep)顶部井神经基底媒体(NBM)

注:该设备可以孵育过夜和镀细胞第二天,或电镀前的细胞培养与NMB +因素至少3小时。

准备装载到设备的神经元

我们使用18日龄胎鼠大脑皮层,我们可以称为脑位或在这里为我们准备在校园的公司购买。我们希望得到组织的新鲜。

  1. 获得1个胎儿大脑皮质组织(2件)存储在休眠Ë
  2. 以3个长玻璃滴管,并先后火成较小的尺寸每个使用酒精灯
  3. 删除从休眠发送一个玻璃吸管在2毫升的NMB因素在无菌15毫升管组织。
  4. Trituration:使用一个灯泡和玻璃吸管皮层是通过向上和向下约5至10倍。然后,下一个较小的吸管,用吸管向上和向下的组织。最后,最小的吸管,用吸管向上和向下的组织。我们要确保有不可见的大块。

    注:近日,全度妍实验室开始比较存储在Hibernate发送准备从最初在50%与0.125%胰蛋白酶处理的组织细胞,组织细胞CA +自由和Mg + +免费清扫缓冲区。的共识是准备使用胰蛋白酶产量比组织更可行的最初放置在Hibernate E.如果你不打算马上使用组织的细胞,组织,那么你就需要存储在Hibernate E.组织

    如果你是打算向使用组织马上和想提高生存能力,然后用胰蛋白酶处理组织机械trituration如下:1)稀释1毫升的钙+ +免费镁+ +免费1毫升0.25%胰蛋白酶(Gibco公司,Invitrogen公司)夹层缓冲液(最终胰蛋白酶= 0.125%)到15毫升管(保持冰冷); 2)缓冲液中进行组织和孵化立即为8分钟,在37 ° C; 3)添加DMEM/10毫升10%胎牛血清停止胰蛋白酶反应,继续下面的协议。

  5. 在1分钟1100转离心细胞
  6. 送气媒体小心关闭细胞沉淀
  7. 加入1毫升的NMB因素重新悬浮颗粒和它的吹打
  8. 经过重力流通过一个过滤器删除任何团块(屋宇署猎鹰细胞过滤40微米尼龙)重悬细胞
  9. 计数和板块细胞:
    • 到离心管中,加入60μLNMB +因素,再拌入20μL细胞悬液,台盼蓝和20μL
    • 新增约10μL此解决方案的血球计数活细胞(蓝色)
    • 调整浓度为2.5 - 4.5 × 10 6细胞/ ml

      注:我们通常获得浓度约为2.5 - 4.5 × 106细胞/ ml。根据卷上的细胞悬浮(通常为1毫升的NBM + B27/Glutamax/PenStrep percortex)。如果组织准备,与胰蛋白酶的产量可能会有所增加。

电镀设备

  1. 装入到一个很好的设备细胞(初级神经元)
  2. 板5μL%的小型设备和大设备每20μL

    注:您可以负载设备的顶部和10μL设备的底部(总量的一半)10μL的细胞,或直接所有的细胞somal以及在顶部的20μL(5μL以及在顶部somal小型设备)。

  3. 孵育10分钟在37℃培养箱中培养,使细胞可以开始重视
  4. 新增约150/30μLNMB +的因素以及对设备的大小取决于每个

    注:卷可能会有所不同厚度的PDMS。重要的是,井已满。

补充在使用无血浆粘合设备

没有等离子治疗,一名技术人员,它的成功没有血浆每周,克里斯蒂娜涂说,只是马上载入细胞。

记住删除SOMA方井(轴突方都不会有问题,如果你离开媒体)媒体。它的流体的流动,使这些细胞进入主渠道。如果你有在水井的媒体,你不会得到流体的流动。

Discussion

在这里,我们展示了如何使用,而不需要等离子体清洁的神经元微流体装置。我们参考这种非等离子的粘接。

Materials

Name Type Company Catalog Number Comments
PDMS Reagent Dow Corning Sylgard 184 with curing agent Please consult Dow Corning to find a vendor near you
Cornig No. 1 Cover Glass Cover Glass Corning Corning No. X2935 244 Available through Fisher Scientific, Fish Catalog number 12-531D
B27 Reagent Invitrogen 17504-044 B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.
Glutamax Reagent Invitrogen 35050-061 Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.
60 mm Petri Dish Tool Fisher Scientific 08-757-13A 60 mm polystyrene sterile petri dishes are used to house the device bound to glass.
Poly-L-Lysine Reagent Sigma-Aldrich P-1274
BD Falcon 50 ml Tube Tool BD Biosciences Falcon No. 352098 Available through Fisher Scientific catalog number 14-959-49A
BD Falcon 15 ml tube Tool BD Biosciences Falcon No. 352097 Available through Fisher Scientific catalog number 14-959-70C

DOWNLOAD MATERIALS LIST

References

  1. Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. Microfluidic culture platform for neuroscience research. Nat Protoc. 1, (4), 2128-2136 (2006).
  2. Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. 2, (8), 599-605 (2005).

Comments

14 Comments

  1. can't view videos?? firefox latest plugins???

    Reply
    Posted by: Anonymous
    November 4, 2007 - 10:33 PM
  2. Hi Ron. I am sorry you are having trouble. To view videos, please make sure that you have the latest version of Flash installed. If you still have problems, please send us an email to support@jove.com with operating system and browser information.

    Reply
    Posted by: Anonymous
    November 5, 2007 - 12:48 AM
  3. What protocol do you use when making the PLL solution? 5mg of PLL to 50ml water?

    Reply
    Posted by: Anonymous
    April 7, 2008 - 2:47 PM
  4. Hi Lee T
    We dissolve the PLL in borate buffer
    1.²4 g of boric acid and 1.9 g of sodium tetraborate is added to 300 ml of nanopure water (final volume will be 400 ml) and stirred to disolve for about 30 minutes.  Next the pH is adjusted to pH 8.5.  ²00 mg of PLL is added and stirred until disolved.  The final volume is adjusted to 400 ml.  The PLL solution is then sterile filtered and aliquoted into sterile 50 ml tubes in a tissure cutlure hood.  Aliquots are stored at -²0C until use.
    Joseph Harris

    Reply
    Posted by: Anonymous
    May 7, 2008 - 5:57 PM
  5. Just wanted to let everyone know a company is forming to make, sell, and distribute the devices. Email xonamicrofluidics@gmail.com for more information.

    Reply
    Posted by: Anonymous
    July 4, 2008 - 1:01 PM
  6. Hi there, it it possible to get a protocol for fixation and staining of neurons in microfluidic chambers? Which method is better for this purpose (fixation and staining), plasma- or non-plasma bonding? Is it possible to remove the microfluidic device without damaging the neurons or dŒs it have to stay on during the procedure? By the way: A really impressive method, fantastic! Looking forward to your answer. Best, Harry

    Reply
    Posted by: Anonymous
    November 6, 2008 - 2:32 PM
  7. Hello Harry Probably the best thing would be for you to check out our company website at http://xonamicrofluidics.com/ We have a protocol there available for download.  It gŒs over staining in the device. Also, if you would like to discuss staining in more detail or have any other questions feel free to email us xonamicrofluidics@gmail.com

    Reply
    Posted by: Anonymous
    November 10, 2008 - 4:46 AM
  8. Hi Harry just to follow up.. The device dŒs not have to be removed for staining. However, if you are interested in visualizing in the mid-region of the grooves then it might not stain very well with the device on.  If you only want to visualize neurons in the main channels or axons just entering or leaving the grooves then staining with the device on is just fine. If you want to get a good stain of the middle of the microgroove region then it is best to remove the device before staining.  This means non-plasma bonding will have to be done as plasma bonding is irreversible. Yes, removing the device can and dŒs damage the neurons.  However, done with care and after some practice it is possible to remove the device and keep most of the sample intact. Some researchers like to fix the cells in the device, place the device on ice for 5 minutes then carefully remove it.  Others just remove the device as carefully as possible prior to staining. Please visit our website http://xonamicrofluidics.com/ for our detailed protocol. Also feel free to email us xonamicrofluidics@gmail.com

    Reply
    Posted by: Anonymous
    November 10, 2008 - 4:55 AM
  9. Hello JŒ, thank you very much for your reply and support. The protocol on http://xonamicrofluidics.com/ which you are refering to is indeed very detailed and helpful. I am now curious to see how my experiments work out. :-) All the best and thanks again, Harry

    Reply
    Posted by: Anonymous
    November 11, 2008 - 5:59 PM
  10. This probably dŒsn't work for non-plasma PDMS-PDMS bonding... or, is there a way to treat one of the PDMS layers as glass?

    Reply
    Posted by: Bryn G.
    September 23, 2009 - 8:05 PM
  11. Hello,

    This was a very good video indeed. Could you please let me know how did you make the wells on the PDMS. They are not micronsized wells that we used to make by replicating form a potolithographed Silicon wafer. Did you use some kind of belt puncher? What is the exact product number and brand of the puncher? I use one to make mm sized well on the PDMS slam, however the edges are not good. Or did you use other technique to make such nicely sheped wells? If I want to make recangular or square shaped wells (² mm length/width and .5-1.0 mm height) on PDMS what could I do? I will appreciate your kind advices.

    Reply
    Posted by: Anonymous
    March 31, 2012 - 6:39 PM
  12. Hello Taifur

    The wells are 8 mm in diameter. We have special punches machined to punch the wells. However, you can use 8 mm biopsy punches made by Sklar. VWR or Fisher Sci should have them.

    Reply
    Posted by: Joseph H.
    April 6, 2012 - 12:48 PM
  13. For the cells you receive in Hibernate E from Brain Bits, do you digest them with trypsin or even digest them at all prior to trituration. Why or why not?

    Thanks.

    Reply
    Posted by: Angela D.
    July 4, 2012 - 10:08 PM
  14. For the cells you receive in Hibernate E from Brain Bits, do you digest them with trypsin or even digest them at all prior to trituration. Why or why not?

    Thanks.

    Reply
    Posted by: Angela D.
    July 4, 2012 - 10:10 PM

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