Damlacık Temelli Bir Mikroakışkan Sistemde Akış Koşullarının Hücre Enkapsülasyonu için Optimizasyonu ve Hücre Dağılımının Stokastik Analizi

Abstract

Detailed analysis of cellular responses is crucial for the evaluation of drugs and therapeutic methods. Droplets generated using two-phase microfluidic systems can be considered as uniform miniature bioreactors that enable three-dimensional (3D) culture. Predicting the number of cells within a droplet is critical for determining spheroid size in 3D culture studies. The aim of this study was to optimize the flow conditions and model the variation of droplet size with temperature and flow rate to investigate the effects of different cell types and initial cell concentrations on droplet size and the number of cells within the droplet. A flow-focusing system with a 100 μm junction width and biocompatible soybean oil as the continuous phase was used to produce droplets with a maximum diameter of 100 μm. Water, DMEM, MEM+10%FBS for MC3T3-E1 cell line, and RPMI+10%FBS for L929 cell line were used as the dispersed phase for comparative analysis of flow and droplet formation processes. The ranges of two-phase flow regime were determined for these systems based on the continuous and dispersed phase flow rates, Qc and Qd, and the capillary number, Ca. The droplet flow regime was found for water at 0.05≤Qd/Qc≤0.25 and 0.09≤Ca≤0.002 and for other biological fluids at 0.05≤Qd/Qc≤0.5 and 0.1≤Ca≤0.001. The design Qd/Qc ratio of 0.05 enabled the attainment of the targeted droplet sizes for all fluids. For culture mediums, the longest inter-droplet distance was obtained with Qd=0.4 μl/min and Qc=8 μl/min and the frequency was calculated as 15 droplets/s. The variation of droplet size with temperature and flow rate was modeled using the calculated values of the viscosity of the fluids and iv the interfacial tension between the phases. In the cell encapsulation studies, L929 fibroblast and MC3T3-E1 osteoblast cell lines with sizes of 15-20 μm and 20-30 μm, respectively, were used at concentrations of 1, 5, 7, and 10 million cells/mL. Increasing the initial cell concentration and droplet diameter, increased the average number of cells entering the droplet; however, a lower average number of cells was found inside the droplets for larger cells. Under optimal flow conditions, the probability of capturing three cells per droplet was determined to be the highest for both cell lines. When 5 million L929/mL was used, the number of captured cells varied between 0 and 19 and the probability of capturing three cells was 22.4%. With 7 million MC3T3-E1/mL, the number of captured cells varied between 0 and 13 and the probability of capturing three cells was 19.5%.