Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices

Abstract

Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5–8.5) and pre-incubation at low temperature (∼4 °C) before polymerization at 37 °C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening.

Introduction

It is now well known that cellular function in 2D and 3D systems is considerably different due to the limited interaction between cells and their microenvironment in 2D culture systems [1], [2]. 3D in vitro cellular models provide enhanced interaction not only among cells but also with ECMs, more closely mirroring the morphology and phenotype of cells in vivo. In solid tumors, cancer cells in vivo exist in a 3D tumor mass, thus cancer growth, invasion, and metastasis are mainly governed by the complex interactions between cells and their microenvironment [3], [4]. For instance, Wang et al. has shown that antibodies against β1-Integrin changed the behavior of breast cancer cells in 3D culture but not in 2D culture [5]. Various 3D cell culture systems such as ex-vivo culture, cellular multilayer, hollow-fiber bioreactor, matrix-embedded culture, multicellular tumor spheroid have been developed in attempts to mimic in vivo microenvironment in vitro [6], [7], [8], [9]. Among those, matrix-embedded culture is a widely used method both in micro and macro systems due to its simplicity and versatility, and thus, is our focus.

The ECM consists of many different polymers. Collagen type I is one of the most abundant polymers in ECMs in vivo, and it is widely used for both micro and macro scale 3D cell culture. Because of its hierarchical structure, the physical properties of collagen are influenced by polymerization conditions such as pH, temperature, and polymerization rate [10], [11], [12]. Collagen molecules are mostly acid-soluble, consisting of homogeneous collection of thin rod shaped molecules (∼1.5 nm wide, ∼300 nm long) before polymerization, and they generate heterogeneous cross-linked structures when the conditions are adjusted to near physiological values (i.e. pH of 6.5–8.5 and temperature of 20–37 °C). Collagen polymerization goes through two phases: a nucleation phase during which molecular assembly occurs, and a rapid growth phase during which cross-linking takes place. The final thickness of collagen fibers (>200 nm wide) is determined during the nucleation phase, where lower pH and lower curing temperature provide a longer nucleation phase, generating thicker collagen fibers [13], [14]. Because collagen is widely used and its polymerization process well understood, we have chosen to focus on collagen for this study.

From a technology perspective, the miniaturization of 3D culture systems holds the promise of enhanced efficiency and functionality. As numerous factors are involved in stimulating or inhibiting cross-talk between cells and their microenvironment, the use of microsystems may be beneficial to examine the factors with less required time, effort, and sample. For example, an adaptable hydrogel array for 3D cell culture has been realized using microfabricated multiwells to study the influence of various ECM parameters on cell behavior with enhanced throughput [15]. In addition, unique geometries and structures have been created in microsystems, mimicking 3D tissues in vivo. For example, using microfluidic patterning and contraction of biopolymers, Tan et al. have constructed two- and three-layer cell-matrix structures that mimics in vivo tissue such as blood vessels [16]. Additionally, 3D in vitro hepatic tissue models and microfluidic scaffolds have been established by incorporating improved microflow control within complex 3D structures [17], [18], [19]. Physical properties of microflow (e.g. laminar flow) have also been employed to partition and compartmentalize 3D co-culture systems [20]. Although these novel techniques improve functionality over conventional 3D systems, their operational difficulty and poor reproducibility limits practical use. Thus, there is a need for more robust and high throughput 3D culture methods. Microfluidics has the potential to fill that need. However, it is important to consider the inherent physical differences between microchannels and canonical open well formats (e.g. surface area to volume ratio, small volumes, materials) and how these differences influence the resultant ECM characteristics. It is only by understanding the interplay between the physics of the microscale and the polymerization process, that one can develop an optimal process for microchannel 3D culture.

We have noted that the condition-dependent manner of collagen polymerization becomes more pronounced in microsystems due to surface area to volume ratio, volume, and material. To construct a reliable 3D culture platform for a broad range of applications, reproducibility of the base culture system is essential. Therefore, in this work, we have investigated various parameters involved in collagen polymerization in microchannels and characterized the polymerization process to enhance reproducibility of the system. Human mammary fibroblast (HMF) cells are used as a model cell type because, based on our initial experimental evidence, they are susceptible to the mechanical properties of the 3D collagen matrix. The length of the nucleation phase of polymerization is known to be one of the critical determinants of fiber diameter. We control the nucleation phase by varying temperature and pH. The large surface area to volume ratio of a microchannel leads to more rapid temperature changes and faster termination of the nucleation phase. Therefore, the gel condition before warming is determinative of final structure after polymerization. Array-based microchannels are used to efficiently investigate the process parameters. Viability tests and stress fiber analysis of HMF cells are performed to measure cellular responses to the 3D microenvironment.