Высокая пропускная способность Single-камеру и несколько клеток микро-инкапсуляции

Summary

Объединение монодисперсных падение поколения с инерционным порядком клеток и частиц, описывается метод инкапсуляции нужное количество клеток или частиц в одной капле в кГц ставки. Мы демонстрируем эффективность в два раза превышающие неупорядоченных инкапсуляции для одно-и двух-частиц падает.

Abstract

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency P_k=1 of 83.7% and a double-particle encapsulation efficiency P_k=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed.

Introduction

Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.^1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.³ Encapsulation of cells in drops has been utilized to improve detection of protein expression,⁴ antibodies,^5,6 enzymes,⁷ and metabolic activity⁸ for high throughput screening, and could be used to improve high throughput cytometry.⁹ Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry¹⁰ and targeted surface cell coatings.¹¹ Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation¹² which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.

To achieve monodisperse drop generation, microfluidic “flow focusing” enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.¹³ For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Q_oil and Q_aq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.¹⁴

When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops D_k containing k cells is dictated by Poisson statistics, where D_k = λ^k exp(-λ)/(k!) and λ is the average number of cells per drop. The fraction of cells which end up in the “correctly” encapsulated drops is calculated using P_k = (k x D_k)/Σ(k’ x D_k‘). The subtle difference between the two metrics is that D_k relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and P_k relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low λ) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric P_k would be high, the majority of drops would be empty (low D_k), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.¹⁵

Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg¹⁶ and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).^17,18 Here, the Reynolds number Re =uD_h/ν and particle Reynolds number Rep =Re(a/D_h)², where u is a characteristic flow velocity, D_h [=2wh/(w+h)] is the hydraulic diameter, ν is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Re_p increase. Note that the high Re and Re_p requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.¹² However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.

Because very few studies address inter-particle train spacing,^19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,¹² a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.^4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.¹²

In previous controlled encapsulation work,¹² the average number of particles per drop λ was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when λ is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Protocol

Протоколы в этом разделе описываются материалов и оборудования, используемого в частности получить экспериментальные результаты. Обратите внимание, что альтернативные поставщики для химикатов и оборудования могут быть использованы. 1. Изготовление устройств и мягкие ?…

Discussion

Несмотря на относительно высокую степень упорядоченности, не все капли будет содержать нужное количество частиц или клеток. Инкапсуляции эффективность может рассчитываться как число клеток или частиц, которые становятся заключены в каплях с желаемой занятости разделить их общего чи…

Materials

Name of the reagent	Company	Catalogue number	Comments
AutoCAD	AutoDesk
Transparency Mask	Fineline Imaging Inc.
SU-8 Photoresist	MicroChem	2050
Dektak Profilometer	Veeco
Petri Dish	BD Falcon	351058
PDMS Silicone Elastomer Kit	Dow Corning Corp.	Sylgard 184, Material Number (240)4019862
Vacuum Desiccator	Jencons	250-030
Vacuum Pump	Alcatel Vacuum Technology	2010 C2
Vacuum Regulator	Cole-Parmer	EW-00910-10
Oven	Thermo Scientific	Lindberg Blue M, OV800F
Biopsy Punch, 0.75 mm	Harris	Uni-Core 15072
Laboratory Corona Treater	Electro-Technic Products Inc.	BD-20AC, SKU 12051A
Glass Slides	Gold Seal	3010
Aquapel	PPG Industries		Alternative Strategy
Polystyrene Microspheres, 9.9 μm	Thermo	G1000
OptiPrep	Sigma-Aldrich	D1556	Not Demonstrated
Luer-Lok Syringes	BD	1 mL: 309628 3 mL: 309585
FC-40 Fluorocarbon Oil	3M Inc.	Sigma Aldrich, F9755
PFPE-PEG Fluorosurfactant	RainDance Technologies
Light Mineral Oil	PTI Process Chemicals	08042-47-5	Alternative Strategy
Mineral Oil Surfactant	Evonik Goldschmidt Corporation	ABIL EM 90	Alternative Strategy
Tygon PVC Tubing	SmallParts	TGY-010
30 Gauge Luer-Lok Syringe Needle, 1/2″	SmallParts	NE-301PL-C
Inverted Microscope	Carl Zeiss Imaging	Axio Observer.Z1
High Speed Camera	Vision Research	Phantom V310
Syringe Pumps (2)	Chemyx Inc.	Nexus 3000
Silicone Oil	Dow Corning	200 fluid, 10 cSt	Optional for Emulsion Storage