Study on chemotaxis and chemokinesis of bone marrow-derived mesenchymal stem cells in hydrogel-based 3D microfluidic devices
© The Author(s). 2016
Received: 26 May 2016
Accepted: 15 July 2016
Published: 2 August 2016
Controlling the fate of mesenchymal stems cells (MSCs) including proliferation, migration and differentiation has recently been studied by many researchers in the tissue engineering field. Especially, recruitment of stem cells to injury sites is the first and crucial step in tissue regeneration. Although significant progress has been made in the chemotactic migration of MSCs, MSC migration in three dimensional environments remains largely unknown. We developed a 3D hydrogel-based microfluidic-device to study the migration behavior of human MSCs in the presence of stromal-cell derived factor-1α (SDF-1α), interleukin 8 (IL-8) and Substance P (SP) which have been utilized as chemoattractant candidates of human mesenchymal stem cells (hMSCs).
We systematically investigated the chemotactic migration behaviors of hMSCs and their responses to SDF-1α, IL-8, and SP. SDF-1α was shown to be the most fascinating chemoattractant candidate among those factors at a certain time point. We also found that each chemokine showed different chemoattractant abilities according to their concentration. In the case of SP, this factor showed chemokinesis not chemotaxis. Especially at a 7–8 × 10−8 M concentration range, the chemokinesis ability driven by SP was further increased. The data suggest that some factors at the optimal concentration exhibit chemokinesis or chemotaxis in a 3D hydrogel-based microfluidic device.
In this study on chemotaxis and chemokinesis of hMSCs, the system parameters such as chemokine concentration, system stability, and 2D or 3D microenvironment are critically important to obtain meaningful results.
Chemotactic behavior is a characteristic of various cell types engaging in biological processes such as inflammation, wound repair, organ development, neurite outgrowth, and tumor invasion . A chemoattractant is defined as a chemical agent that induces cell migration toward itself. This agent includes members of the growth factors, cytokines and chemokines . Chemotaxis in cells is the movement of cells toward or away from a chemical reagent. Attracted cells exhibit positive chemotaxis while repelled cells exhibit negative chemotaxis. While chemotaxis is a directional behavior, chemokinesis is the random movement of cells. Both endogenous and exogenous substances act as chemoattractants. Therefore, the harmony between endogenous stem cell recruitment and exogenous stem cell induction is one of the most critical issues for effective regenerative therapies in tissue engineering. Many researchers in the tissue engineering field have studied many kinds of chemokines or growth factors to recruit mesenchymal stems cells (MSCs) endogenously. MSCs have closely been involved in the process of healing, and their recruitment to the target area is crucial to enhance their therapeutic effect in the patients. The ability of MSCs to produce juxtacrine or paracrine factors is very important to induce regeneration from the endogenous stem cells. Studies have been done previously on some cytokines that affect the migration of MSCs to injury sites [3, 4]. There are some important cues that should be controlled such as the stemness of the MSCs, culture conditions, and delivery method to induce MSC migration . Some chemokines and growth factors are known to promote selectively proliferation, migration and differentiation of MSCs [6, 7]. For instance, stromal cell-derived factor-1 α (SDF-1α) mediates cell migration by binding with CXC chemokine receptor-4 (CXCR4) at the site of injury [8–10]. However, most results previously have reported limitations because those are based on a two dimensional (2D) culture. It is well known that cells cultured using traditional 2D tissue culture methods are morphologically different from cells in humans or animals.
Our body has 3 dimensional (3D) structures. The cells compromising each organ interact with other cells or circumstances; thus, microenvironments affect cells significantly. 2D cell cultures are unable to perfectly mimic real cell microenvironments and cannot effectively study cell-cell and cell-extracellular matrix (ECM) interactions. In reality, all cells and tissues in vivo or in clinical condition are placed in 3D microenvironments, and some data from in vivo and clinical research done in 3D conditions show discrepancies with the data obtained from 2D in vitro conditions. Signals in 3D environments have a key role in cell differentiation, proliferation and migration of cells. A study on a 3D microenvironment is considered similar to the in vivo environment rather than a 2D culture which lacks, reduces or compromises important signaling events [11, 12]. Due to the limitations of 2D cell culture, 3D cell studies using microfluidic devices have greatly received attention enabling one to assay behaviors of stem cells in a controlled microenvironment with spatiotemporal conditions of the factors. There are some benefits such as a low volume of reagents, fast response time, consistent fluid flow on microscale dimensions of the concentration gradient in microfluidics [13, 14]. Therefore, 3D cell culture platforms are useful tools for mimicking the microenvironment of cells and tissues compared to 2D cell cultures. A number of 3D microfluidic models have been used to study the migration of neural stem cells (NSCs) [15, 16], leukocytes  and tumor cells . Generation of a concentration gradient of cytokines or growth factors inducing single cell responses enable one to characterize the behavior of the cells quantitatively .
In this study, we investigated the chemotactic migration of human bone-marrow derived mesenchymal stem cells (hMSCs) with hydrogel-based microfluidic platforms. To control the conditions, we considered the composition of the hydrogels and microfluidic platform systems. Hydrogels are efficient devices to study chemokine gradient effects to quantify hMSC behaviors. The chemotaxis of hMSCs in microfluidic devices follows a stable gradient. Using a 3D microfluidic system, we studied the chemotactic migration behaviors of hMSCs and their responses to chemoattractants in a 3D microenvironment. Three candidates of chemoattractants, SDF-1α, Interleukin-8 (IL-8), and Substance P (SP) were investigated. Furthermore, we determined the optimum concentration for hMSC chemotaxis.
Microfluidic device fabrication
Cell culture and cell seeding
Human Bone marrow-derived mesenchymal stem cells (hBM-MSCs) were commercially obtained from Severance Hospital. The hMSCs of passage 6 to 8 were cultured in growth medium prepared with DMEM –low glucose (Gibco) with 10 % (v/v) FBS (Welgene) and 1 % (v/v) antibiotics. Cells were maintained in culture and used up to the 7th passage. All cultures were kept in a humidified atmosphere at 37 °C and 5 % CO2. When needed, cells were trypsinized by standard protocols and washed in PBS. Before seeding, hMSCs at passage 7 were suspended at 2.5 × 105 cells/mL in hydrogels. The hydrogels were composed of collagen (collagen type, BD Bioscience) gel (2 mg/ml) and growth-factor reduced Matrigel (GFR-Matrigel) at a 1:1 ratio (Young’s modulus: 9 kPa). The gel channel was filled with the cell suspension to complete the seeding. The gelation process occurred within 30 min in humidity boxes incubated at 37 °C and 5 % CO2. After gelation, the growth medium was injected into both side channels, and the gel-microfluidics were incubated in an incubator for over 24 h.
Chemotaxis and chemokinesis assays
In the microfluidic device, hMSCs in the hydrogel were cultured overnight. After cell spreading, one of the side channels was treated with the chemokine at the relevant concentrations, and the other side of the channel was filled with blank medium. To find out the optimal concentration, each factor was screened for concentrations ranging from 10−6 M to 10−10 M. The candidates of chemokines used in this study were SDF-1α (Peprotech), IL-8 (R&D) and SP (Calbiochem).
Cell tracking and statistical analysis
Images were taken every 10 min. for 24 h with a live cell microscope (Carl Zeiss Axio Observer. Z1) incubated at 37 °C and 5 % CO2. Images from the first 3 h were not analyzed until the concentration gradient of the channels was stable. In the data analysis, cells in both side sections of the central channel were not counted, and cells in section ② in Fig. 1a were analyzed. The central channel of the microfluidic device is divided by three sections, and each section has a 300 um length and a 150 um depth shown in Fig. 1a. Human MSC migration was analyzed with MTrackJ, a manual cell-tracking plugin for the NIH ImageJ software. When the cell tracking analysis was done, cells in section ② were tracked every 20 min for 10 h. Compass plots of cell tracks and angular histograms were quantified from position data using the Chemotaxis Tool plugin (ibidi, Germany). The variations in directional distance along the X-axis (ΔX) were analyzed with the Statistical Package for Social Sciences (SPSS).
Results & discussion
Microfluidic device identifies chemotactic migration showing stable gradients
Chemokinesis of hMSCs on a chemokine gradient
To assess chemoattractant migration of hMSCs, we used the 3D microfluidic system which was designed based on the Singapore model originally developed at MIT-KU [16, 20]. One of the side channels in the microfluidic device was filled with cytokines, and the other side was filled with blank medium without cytokines. The middle channel of the device was filled with the hydrogel containing hMSC cells (Fig. 1).
The chemotactic response over time
In this study, we systematically investigated the chemotactic migration behaviors of hMSCs and their response to SDF-1α, IL-8 and SP. SDF-1α is one of the most fascinating chemoattractant candidates at certain time points among the factors tested in this study. We also found that each chemokine exhibited a different chemoattractant ability according to its concentration. Chemokines and growth factors in previous reports induce hMSCs migration towards a high concentration which is known as chemotaxis; however, we did not observed any noticeable chemotactic behaviors in MSCs. This discrepancy between our results and the results reported by other groups might be due to the system conditions (2D vs 3D).
BMSCs, bone marrow stromal cells; CXCR4, CXC chemokine receptor-4; ECM, extracellular matrix; hBM-MSCs, human bone marrow-derived mesenchymal stem cells; hMSCs, human mesenchymal stems cells; IL-8, interleukin-8; MSC, mesenchymal stems cell; NSC, neural stem cell; SDF-1α, stromal cell-derived factor-1 α; SP, substance P; SPSS, statistical package for the social sciences
We specially thank Dr. Sewoon Han at Korea University for professional advice to prepare microfluidic devices.
This work was supported by the research resettlement fund for the new faculty of Seoul National University, invitation program for distinguished scholar, and a grant from the Korea Institute of Science and technology (KIST, 2E24340).
Availability of data and materials
Further data shall not be shared because patent application is in progress.
KL, CHA, DY and HJ developed the concept and designed experiments. DY, EL and MHP performed the whole process of experiments. SC provided the 3D microfluidic device. KL, DY and HK extensively contributed on the manuscript preparation. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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