3D胶原凝胶和微通道3D相互作用的研究准备<em>在体内</em

Summary

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。

Abstract

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.

Introduction

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.

Protocol

1.中和，稀释和3D调查和细胞收缩测定胶原蛋白溶液聚合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。 稀释中和胶原蛋白与细胞介质适当浓度（如RPMI，DMEM 等 ）。 来计算，以弥补所需胶原浓度所需中和胶原?…

Representative Results

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?…

Discussion

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，?…

Materials

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 .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	2ml, 5ml, and 25ml
70%EtOH	Fisher	NC9663244