3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

摘要

胶原是细胞外基质的核心组件，并提供必要的暗示了几个细胞过程，从移植到分化和增殖。这里提供了一种用于三维胶原凝胶内嵌入电池的协议，和用于产生使用的PDMS微通道的随机或对齐胶原基质的更先进的技术。

摘要

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis¹. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration^2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

引言

Many cellular processes have been extensively studied in 2 dimensions, thereby forming a collective knowledge of basic cell signaling mechanisms. These studies, however, neglect important cellular cues received from the structural and mechanical properties of the local cellular microenvironment and extracellular matrix (ECM). To better understand how cells interact within a physiological context, it is important to study them in 3-dimensional (3D) assays. The ECM for these 3D assays can either be cell-derived or reconstituted from purified proteins. Regardless of the source of the ECM, 3D matrix assays have proven to be invaluable for understanding how cells navigate and interact within the physiological world. For example, cells grown in 3D matrices display distinct modes of locomotion that depend on the mechanical nature of their surrounding ECM which are not observed in 2D experiments^4-6. Moreover, cells cultured in 3D also have fewer and less pronounced stress fibers and focal adhesions than their counterparts grown on hard surfaces such as glass or plastic⁷.

The importance of contextual 3D assays is not limited to cell migration, however. Some other cell signaling events can only be investigated through the use of 3D assays. During tissue and cell differentiation, the stiffness of the extracellular environment and ECM provides signals that can influence morphogenic events. For example, mammary epithelial tubulogenesis only occurs in low stiffness 3D matrices, but not in stiff matrices nor on 2D substrata^8,9. When cultured within stiff 3D matrices, these same epithelial cells take on an aberrant phenotype with increased proliferation and cell membrane protrusions driven through altered FAK and ERK signaling¹⁰. Many other signaling pathways and cellular processes are known to be similarly affected by the stiffness of the local cellular environment, and these signaling cascades highlight the importance of investigating signaling events and cellular phenotype in the context of appropriate local mechanical properties of a 3D ECM.

Collagen I is a particularly relevant protein to use for in vitro studies as it is the most abundant component of the ECM and is responsible for many of the mechanical properties of the cellular microenvironment. While it was originally thought of as merely a structural protein, its role is now known to be much more complex. Collagen fiber composition, architecture, orientation, density, and stiffness all provide a concentrated milieu of signaling information⁵. During the progression of certain diseases, such as chronic inflammation and tumorigenesis, the collagen network is dramatically remodeled^2,11. More specifically in breast cancers, increased collagen deposition and tissue stiffness accompany and likely contribute to tumor progression. In these early tumors, the stiffened collagen network appears strained and more aligned, such that most of the fibers encapsulate the growing tumor². As the tumor progresses, the collagen continues to reorganize, and regions of the fibrillar network become orientated perpendicular to the tumor boundary^2,12. Perpendicular alignment serves as a prognostic biomarker where these patients have a poorer disease free progression and overall survival¹. One explanation for this correlation is that the poor outcomes are a consequence of increased dissemination of tumor cells via persistent cell migration in aligned collagen networks³.

To understand how cells specifically respond to alignment and organization that is observed in tumor progression, it is necessary to generate both random and aligned 3D collagen matrices for experimentation. There are three basic methodologies to induce alignment within fibrillar networks. The first technique utilizes a strain-inducing device where the collagen between two points is contracted or stretched to generate alignment. Fibers parallel to the axis of force are pulled taut while fibers perpendicular to the axis are compressed and buckled. While strain-induced techniques typically offer superb alignment, this approach requires bulky equipment that is not easily adaptable to many platforms^3,13. Alternatively, cell-induced strain can be created by placing localized plugs of cells that subsequently contract and align the collagen¹³. This method has the problem of being variable, as many parameters may be subject to change. The second method utilizes magnetic beads and a magnetic field during polymerization to induce collagen alignment^13,14. Good results can be obtained from this method with unsophisticated equipment, but it does require the use of antibodies or some other method to magnetize the polymer. Therefore, it can be somewhat expensive to use, and the stiffness of the collagen gel is potentially modified by the increased connections in the network. Moreover, the magnetic beads used in this process are often autofluorescent, which is problematic for imaging experiments. Lastly, alignment can be generated by PDMS microfluidic channels^3,15,16. In this method, collagen alignment is achieved by flowing polymerizing collagen through small microfluidic channels. These microfluidic channels can be made in a multitude of designs, and are easily adaptable to many platforms. Moreover, they are very economical as very small quantities of collagen and other reagents are used due to their diminutive sizes.

Provided here is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. In addition, a more advanced technique, wherein PDMS microfluidic channels are used to control the organization and alignment of the collagen matrix is also provided. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a 3-dimensional, physiological microenvironment.

研究方案

1.中和，稀释和3D调查和细胞收缩测定胶原蛋白溶液聚合

1. 在无菌组织培养罩冰，中和胶原（1:1）用无菌，冰冷的100mM HEPES在2×PBS中，pH为7.4，在15ml锥形管中。用塑料吸管调匀直至溶液均匀混合漩涡不再可见。要小心，不要在混合过程中引入气泡。简单地存放在冰上。
2. 稀释中和胶原蛋白与细胞介质适当浓度（如RPMI，DMEM 等 ）。
   1. 来计算，以弥补所需胶原浓度所需中和胶原蛋白的量，使用等式N =（D * V）/（S / 2），其中N为所需中和胶原蛋白的量，D是所需的胶原浓度，V是所需胶原浓度的最终体积，以及S是叔他首发股票的胶原蛋白浓度。
   2. 通过加入细胞和细胞培养基溶液的中和胶原的量带来补足体积。调匀并在冰上储存直至准备施法。
      1. 例如:对于在1ml凝胶体积有2毫克/毫升凝胶（典型体积为在6孔板或50毫米的玻璃底皿的凝胶浇铸）中，首先通过将所需最终体积乘以所希望的浓度（2毫克/毫升） （1毫升）中。借此号码（2毫克）和由瓶列出的原料浓度（9.49毫克/毫升）的一半除以它。在这种情况下，0.42毫升中和胶原蛋白与0.58毫升介质/细胞混合物的稀释。
3. 吸管冰冷胶原/细胞/培养基混合物变成一个非组织培养物处理的6孔板或50毫米的玻璃底培养皿中。用枪头均匀摊开的解决方案。
   注意:使用非组织培养处理板向细胞的附着或生长减少collag之外是很重要恩凝胶。
4. 允许在室温下聚合大约10 - 15分钟。凝胶应当在聚合变成不透明的。
5. 它已经变成不透明后，移动板或盘，以37℃额外45 - 60分钟以完成聚合。
6. 经过45 - 通过运行以及周围或盘周边P200的枪头从井两侧的3ml媒体和释放凝胶 - 60分钟，加2。漩涡菜轻轻地释放凝胶。胶原凝胶应该在媒体上浮动。

2.免疫印迹，细胞形态和凝胶含量Gontractility

1. 评估与蛋白质水平，形态或细胞收缩到ECM刚度，浇胶原凝胶开始，按照1.1 - 1.6，与细胞的接种7 - 10天检测。根据增长率和融合凭经验确定每个细胞系播种量。为10天的试验中，接种密度的范围可以从20,000 - 100,000个细胞/凝胶。
2. 测量细胞收缩，通过使用尺子或照相机每24小时或在适当的时间间隔测量的凝胶直径。
   注意:另外，从显微镜收集对应的图像可以检查在凝胶接种，如腺泡状结构，上皮小管，蜂窝突起，和lameliapodia细胞系的形态特征的特征。
3. 为了评估蛋白质水平，如在沃兹尼亚克等人 ¹⁷和Gallagher 等人 ¹⁸所述裂解在RIPA凝胶缓冲液和处理为感兴趣的蛋白质的Western印迹分析。
   1. 重要信息:在细胞系之间的收缩的比较，正常化收缩到总DNA，其可从凝胶中提取由吕所概述， 等人 ^19，或到一个不变，管家蛋白（组蛋白，GAPDH 等 ）通过Western。印迹分析，如在沃兹尼亚克等人 ¹⁷和Gallagher 等人所述^。18。如果凝胶内的细胞计数使用血球，确保正常化细胞数占总凝胶面积承包凝胶将集中细胞。
4. 4天通过除去将1ml培养基并用1ml新鲜培养基更换它 - 每3饲料凝胶。确保测量之后的加入新鲜的媒体/血清会引起收缩穗养活凝胶。

胶原蛋白光纤对准PDMS微通道3代

注:要对齐产生胶原基质，用于PDMS微通道（ 图 2A）的模具，需要通过软光刻¹⁵取得了SU-8硅主。

1. 为了使PDMS通道，彻底的一次性​​杯具一门手艺棒混合PDMS。对于一个6英寸的硅主人，用2克固化剂混用20克橡胶基地。
2. 解气体的PDMS混合物通过的真空压力下放置一次性杯在真空室550毫米汞柱。解气体为1 - 1.5小时。
3. 虽然脱气的PDMS，准备硅主浇注。通过将透明胶片丢球到烤盘其次是有机硅主做准备。确保微模具朝上。
   注:在PDMS浇铸和固化，硅氧烷主将透明膜的两片之间夹入。
4. 后1 - 1.5小时，从真空室取出脱气的PDMS，慢慢倒在硅氧烷主。
   重要提示:避免气泡。继续主中心纷至沓来，让重力均匀地传播它。
   注:PDMS下降并不需要一直延伸到主的边缘。
5. PDMS已经被倾倒在主后，对硅主的PDMS上应用投影胶片的第二片。小心地倒在PDMS /硅胶高手顶部滚动第二透明薄片，以避免产生气泡。不着急。 PDMS应均匀地散布现在Ø版本高手。
6. 轻轻地放在透明胶片上的橡胶片，随后通过1/8"丙烯酸系片材。
7. 在压克力板顶部添加3个10磅的重量。最初，权重将"浮动"。让他们进一步处理之前解决和稳定。
8. 设置加热板的温度至70℃，固化的PDMS 4小时。允许干扰前冷却1小时的额外最低。
9. 小心地从晶片剥离透明膜的顶片，并用钳子取出频道。
10. 存放在无尘的菜，直到准备使用。

4. PDMS预学习的微通道使用

1. 地方频道上新的，干净的透明胶片倒挂。清洁所有端 ​​口（ 图2b），使用打圈用锋利的钳子。删除PDMS中的任何片段。
2. 清洁通道使用了一块打包带作为钉布。将胶带粘工作台表面（粘面朝上），然后设置通道0■首页。向下按压钳的圆端渠道，以确保良好的接触。删除，直到可见的碎屑被去除两侧重复。
3. 转移清洗，坦然PDMS渠道到50毫升锥形用70％乙醇。在涡旋30秒的最大速度。丢弃EtOH中并用新鲜的70％乙醇取代。储存在70％乙醇中待用。
4. 在组织培养罩，并使用无菌技术，传输PDMS渠道，一个洁净无菌盖玻璃或玻璃底菜。把通道侧和紫外线治疗，直到乙醇蒸发。
5. 干燥后，翻转PDMS所以渠道都面临着向下延伸玻璃罩。按PDMS通道下放到玻璃板做一个良好的密封。添加无菌的PDMS覆盖的贴剂/关闭中心端口（端口B， 图2b）。允许继续之前彻底干燥。
6. 预涂层用无菌水10微克/毫升的胶原蛋白的信道的内侧。大衣，放置一个100微升滴在陈荫罴顶部埃尔，并用真空抽吸通过。后在37℃1小时，填充有胶原涂层溶液的冰箱传输通道。冷藏约15 - 30分钟。
7. 用吸气器或吸移管除去胶原涂布溶液，并开始胶原制备。

5.胶原使用准备在微通道

1. 在冰上，中和胶原（1:1）用冰冷的100mM HEPES在2×PBS中，pH值7.4。彻底混合直到均匀（有关详细信息，请参见1.1节）。
2. 稀释中和胶原蛋白的适当浓度与细胞介质（有关详细信息，请参见1.2节）。孵育在冰上15分钟。
3. 同时，冷冻装在冰频道。
   注意:我们的目标是对信道过程中的所有成分，在4℃或以下。胶原的成核温度和时间是到聚合过程中的关键参数，并且可以是用于进一步优化的起始点，如果需要的话。
4. COUNT细胞与在此时间适当接种密度血球和重悬。为了便于计算，悬浮于1 - 3百万个细胞/毫升。 （更多详情，见第2.1节）。
5. 一旦细胞已被计算在内，15分钟过去了，进行胶原蛋白涌出。

6.浇筑对齐和随机胶原蛋白微通道

1. 通过渠道绘图胶原蛋白之前，调整和设定真空压力内嵌真空调节器。真空压力提供了诱导和控制胶原的流速，它决定对准程度的力。
   1. 随机或不对齐的矩阵，可以使用一个宽的通道（3毫米宽×200微米高）用10毫米汞柱或更低的真空压力。
   2. 对于排列矩阵，使用60毫米汞柱或更高的真空压力狭窄的通道（宽1mm×200微米高）。
2. 从冰中取出安装通道和放置在层流H型清洁，消毒面洪水。
3. 迅速开展工作，并装载120 - 150微升中和胶原蛋白的成A口（ 图2b）。
4. 通过将25毫升移液管连接到真空管线以上端c（ 图2b）绘制通过通道胶原。通过在一个单一的，统一运动画胶原。重要信息:对于随机或对齐矩阵，得出胶原慢慢地穿过通道（约0.5 - 每秒1毫米），并停止一旦它到达终点。对于排列矩阵，得出整个胶原蛋白更快速，但是要注意避免产生气泡。
5. 小心地从P200的Pipetman或吸气港口区域去除多余的胶原蛋白。
6. 将无菌PDMS补丁在两个端口A和C.所有端口现在应该覆盖。
7. 经过2 - 3分钟，取出中心PDMS补丁（B口），并添加2 - 3微升细胞（5 - - 10个细胞）进入中心端口。允许部分聚合（转不透明）在室温下再10 - 15分钟。
8. 10后- 15分钟后，转移至37℃另外15 - 30分钟以完成聚合。除去PDMS盖和介质添加根据需要完全覆盖通道和培养的细胞。细胞可以通过除去一个毫升旧媒体的，并用一个毫升新媒体代替它送入。

结果

而三维测定可以胶原凝胶的相同的刚度中完成，改变凝胶硬度可以用来确定细胞将如何在它们的细胞微环境的机械变化。僵硬胶原凝胶被定义为凝胶，其中嵌入的细胞不能在本地收缩周边的胶原蛋白。不同细胞类型的固有收缩是唯一的，并且因此最好是开始用一个简单的收缩曲线建立将被感测为标准，僵硬的胶原浓度。

通过?...

讨论

3D胶原凝胶是一种宝贵的除了我们的工具箱，了解细胞如何解释和自己的局部微环境作出反应。该原稿提供了一个非常基本的协议用于3D胶原基质中嵌入的细胞，并重复地生成具有随机的或排列的胶原纤维基质。两个协议工作作为适应的平台，其中不同的胶原蛋白同种型，交联剂，或其它基质蛋白可能在聚合时加入。这也很容易修改的平台，以评估基因表达和蛋白生产不同的时段和端点。成像，?...

材料

Name	Company	Catalog Number	Comments
High Concentration Rat Tail Collagen	Corning	354249
SylGard184 elastomer kit	Corning	NC9285739	Elastomer for PDMS channels
HEPES	Fisher	BP310	For HEPES neutralization buffer
KCl	Fisher	BP366	For HEPES neutralization buffer
KH2PO4	Fisher	BP362	For HEPES neutralization buffer
Na2HPO4	Fisher	S374	For HEPES neutralization buffer
NaCl	Fisher	BP358	For HEPES neutralization buffer
Levy Improved Neubauer Hemacytometer	Fisher	15170-208	cell counting
6-well non-tissue culture plate	Corning	351146
50 mm glass bottom dish	MatTek	P50g-1.5-30-f
Bel-Art Plastic Vacuum Desiccator	Bel-Art	F4200-2021	Degassing chamber for PDMS
transparency film	3M	pp2950	Plastic film for pouring pdms channels
ThermoScientific CimaRec	ThermoScientific	HP141925	Hot plate for curing PDMS microchannels
Vacuum regulator	Precision Medical	PM3100	Vacuum regulator for collagen microchannels
8" x 8" rubber sheet	Amazon - Rubber-Cal	Silicone - 60A	rubber sheet for pouring PDMS microchannel
8" x 8" x 0.125" acrylic sheet	Amazon	Plexiglass sheets	for pouring PDMS microchannels
10 lb weights	Amazon	CAP Barbell	for pouring PDMS microchannels
15 ml Conical tubes	Fisher	352097
50 ml Conical tubes	Fisher	352098
Plastic pipets	Dot Scientific	229202B, 229206B, and 667225B	2 ml, 5 ml, and 25 ml
70% EtOH	Fisher	NC9663244