Journal of Colloid and Interface Science 547 (2019) 127–135 Contents lists available at ScienceDirect Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis Regular Article Analysis of centrifugal homogenization and its applications for emulsification & mechanical cell lysis Kaustub Singh a, Ankur Gupta b, Abel-John Buchner a, Fatma Ibis a, Joachim W. Pronk c, Daniel Tam a, ⇑ Huseyin Burak Eral a,d, a Process & Energy Department, 3ME Faculty, TU Delft, Leeghwaterstraat 39, Delft, The Netherlands b Department of Mechanical and Aerospace Engineering, Princeton University, NJ, USA c Department of Bionanoscience, Faculty of Applied Sciences, TU Delft, Van der Maasweg 9, Delft, The Netherlands d Van’t Hoff Laboratory for Physical and Colloid Chemistry, Debye Institute, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands graphical abstract ω article info abstract Article history: We detail the analysis of centrifugal homogenization process by a hydrodynamic model and the model- Received 2 December 2018 guided design of a low-cost centrifugal homogenizer. During operation, centrifugal force pushes a mul- Revised 9 March 2019 tiphase solution to be homogenized through a thin nozzle, consequently homogenizing its contents. Accepted 11 March 2019 We demonstrate and assess the homogenization of coarse emulsions into relatively monodisperse emul- Available online 20 March 2019 sions, as well as the application of centrifugal homogenization in the mechanical lysis of mpkCCD mouse kidney cells. To gain insight into the homogenization mechanism, we investigate the dependence of MSC: emulsion droplet size on geometrical parameters, centrifugal acceleration, and dispersed phase viscosity. 00-01 Our experimental results are in qualitative agreement with models predicting the droplet size. 99-00 Furthermore, they indicate that high shear rates kept constant throughout operation produce more Keywords: monodisperse droplets. We show this ideal homogenization condition can be realized through hydrody- Centrifugal emulsification namic model-guided design minimizing transient effects inherent to centrifugal homogenization. Homogenizer Moreover, we achieved power densities comparable to commercial homogenizers by model guided opti- Centrifugation mization of homogenizer design and experimental conditions. Centrifugal homogenization using the pro- Emulsions posed homogenizer design thus offers a low-cost alternative to existing technologies as it is constructed Lysis from off-the-shelf parts (Falcon tubes, syringe, needles) and used with a centrifuge, readily available in standard laboratory environment. Ó 2019 Published by Elsevier Inc. ⇑ Corresponding author. E-mail address: [email protected] (H.B. Eral). URL: http://www.erallab.com (H.B. Eral). https://doi.org/10.1016/j.jcis.2019.03.036 0021-9797/Ó 2019 Published by Elsevier Inc. 128 K. Singh et al. / Journal of Colloid and Interface Science 547 (2019) 127–135 1. Introduction and analytical modelling. Next, we detail the model-guided exper- imental design of the CHD. The influence of centrifugal speed, Emulsification is an essential process in colloid and interface number of passes, dispersed phase viscosity and nozzle size on science [1]. Emulsions have been utilized as templates for self- the droplet size distribution is studied. Moreover, we demonstrate assembly [2,3] and synthesis of tailored materials [4,5]. Emulsion utility of CHD for emulsification and mechanical cell lysis. The nov- stability is intimately related to droplet size distribution dictated elty of our study lies in the development of a hydrodynamic model by the preparation techniques i.e. details of the emulsification pro- providing a physical understanding of centrifugal homogenization. cess (also known as homogenization) [6–8]. Consequently, a funda- Guided by this model, we were able to account for transient effects mental understanding of the process of homogenization and the inherent to centrifugation with important practical consequences mechanisms dictating the droplet size distribution will inform not for centrifugal homogenization, eg. time-dependent liquid column only development of novel materials but also contribute to improv- height and centrifugal speed. We explained the interplay of exper- ing the stability of commonly used emulsions in industrial practice. imental parameters dictating the droplet size distribution, and In a homogenizer, hydrodynamic shear forces break colloidal consequently emulsion stability. Moreover, we reached power scale entities such as droplets, particles, and cells [9–15]. The phar- densities comparable to commercial homogenizers through the maceutical industry uses homogenizers to create micron-sized hydrodynamic model guided design, despite the use of only readily crystals to enhance dissolution rates of active pharmaceutical available and inexpensive lab supplies in its construction. We ingredients [16–18]. In the food industry, homogenizers are believe physical insights drawn from this study will guide future employed to create droplets that carry hydrophobic nutrients optimization of centrifugal homogenization applications. [19,20]. Additionally, homogenizers are used to create personal care products such as creams and lotions [21] even artificial blood 2. Materials and methods cells [22]. The aforementioned applications often involve an emul- sification step, i.e. breakup of oil droplets suspended in water or 2.1. Assembly of the centrifugal homogenization device vice versa [7,23]. For emulsification, typical homogenizer designs include microfluidic homogenizers [24–26], high pressure homog- The basic architecture of the CHD consists of a reservoir within enizers [14,27–30] and ultrasonic homogenizers [23,27,28]. which the pre-emulsion to be homogenized is stored, and a nozzle Though these approaches excel at high-throughput applications through which the emulsion is forced by way of centrifugation (volume rates order liters per hour), they require high capital (Universal 320 R, Hettich lab technologies), as illustrated in investment and are not designed to handle small volumes on the Fig. 1a. Two versions of the CHD device were constructed and order of 1–10 mL. tested: one with a single-diameter reservoir (the ‘‘single-stage Owing to their ability to manipulate small volumes, microflu- device”), and one with an extra, wider, reservoir section (the idic homogenizers have attracted high levels of attention over ‘‘double-stage device”). These two arrangements are shown the past two decades [31]. Microfluidic homogenization, utilizing schematically in Fig. 1b and c, wherein the flow direction is ori- micromanufactured structures such as barbs or nanowires, have ented downwards. Photographs of the assembled single and dou- been successfully demonstrated for emulsification and cell lysis ble CHDs are given in Supplementary Information. The reasoning [32–35]. Furthermore, microfluidic devices where the fluids are behind the double stage design is explained in Section 3. driven by centrifugal forces have shown promise in cell homoge- All parts used in the construction of the device are readily avail- nization in lab-on-chip applications [36,37]. Such microfluidic able in a standard laboratory. The single stage reservoir for the pre- platforms however usually require dedicated manufacturing emulsion is constructed from a 2 mL transparent plastic syringe equipment or trained personnel. purchased with VWR catalog number 613-1629. For the double Centrifugal force has been utilized in combination with stage device, a 20 mL plastic sample bottle serves as the second, microstructured meshes and membranes to generate high shear wider, reservoir section, and is glued to the upper end of the syr- for industrial homogenization applications [38]. Similar techniques inge (see Fig. 1c and supplementary information). have been exploited in synthesis of hydrogels [39–41], blood The nozzle is made from a standard stainless steel fluid dispens- serum separation [42], and mixing of liquids in microfluidic chan- ing needle, purchased from Nordson. Gauges (G) 27, 30 & 32, with nels [43,44]. Centrifugal force coupled with step emulsification in inner diameter d ¼½108; 160; 210 lm, are tested. The length of laminar flow conditions has also been used for digital droplet n these stainless steel needles is always h ¼ 1:4 cm. The needles recombinase polymerase amplification [45,46] and producing high n are attached to the syringe and are secured with a Luer lock internal volume fraction emulsions [47]. However, a model guiding connection. rational choice of experimental parameters across laminar and tur- The apparatus is inserted inside a standard 50 mL Falcon tube bulent flow regimes has, to best of our knowledge, not previously by drilling a hole in the cap of the tube that matches the outer been proposed. Therefore, we propose a hydrodynamic model diameter of the reservoir. The gap between the syringe and the Fal- and a simple experimental setup by eliminating the meshes and con tube is sealed by using Parafilm. The Falcon tube serves as a membrane, and instead simply forcing the mixture of oil, water, collector for the homogenized emulsions. and surfactant through a thin nozzle. Guided by the proposed model, we detail the design of a low-cost centrifugal homogeniza- tion device (CHD) that is able to process volumes on the order of 2.2. Experimental procedure for emulsification 1—10 mL while maintaining shear rates comparable to commercial homogenizers. The proposed CHD can be constructed