Fabrication of synthetic organelles using bottom-up biology

The procedure showcased in this short review is originally based on the research paper “Bottom up Assembly of Functional Intracellular Synthetic Organelles by Droplet-Based Microfluidics” written by Oskar Staufer, Martin Schroter, Ilia Platzman, and Joachim P. Spatz, published in Small 2020, 16, 1906424. It explores three types of Giant Unilamelar Vesicles (GUVs)- based Synthetic Organelles assembly using droplet-based microfluidics equipment and concepts.

Abstract

Bottom-up synthetic biology has the potential to be applied in the study of complex sub-cellular structures through the formation of artificial compartmentalized systems (Synthetic Organelles) that recreate living cell functions and their mechanical and physiological characteristics. In this study, the authours discuss the controlled assembly of synthetic organelles using droplet based microfluidics. Three distinct types of giant unilamelar vesicles (GUVs)-based Synthetic organelles (SOs) are assembled – (1) Synthetic peroxisomes reinforced cellular stress-management, mimicking an organelle with the use of analogous enzymatic modules, (2) The formation of a synthetic endoplasmic reticulum (ER) which is implemented in intercellular calcium signalling and replicates an organelle similar to the host cell using a fundamentally different mechanism and (3), synthetic magnetosomes, not naturally present in the host cell, mimicking the functions of an organelle from other sub-groups of the phylogenetic tree. The microfluidic assembly of such SO’s will set the pace for advancements in intracellular structures implantable into living cells.

Introduction to synthetic organelles

Synthetic organelles (SO) can be described as sub-compartments of artificial cells to replicate the bio-chemical processes that occur within cells. A bottom-up approach to biology is a methodology to simulate detailed biological models from sub-units of data under different physiological conditions. Fabrication of these synthetic organelles with the help of droplet-based microfluidics has completely revolutionized the field of bottom-up biology. ^[1]

Water-in-oil droplet compartments can replicate the cell-like separation between intra- and extra-cellular space.^[2,3] Such synthetic cells can be used for a range of applications like the formation of cell-sized giant unilamelar vesicles (GUVs) and the controlled assembly of organelles. Organelles are considered to be one of the most important indicators of eukaryotic lifeforms. The goal is not only to explore their fundamental operating principles, but to develop synthetic cellular implants able to rescue cells with dysfunctional organelles.^[6]

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Aims and Objectives

The primary objective of this research is to study the formation of various types of SO’s created through droplet-based microfluidics concepts. The purpose for this research paper can be split into three distinct milestones:

• Mimicking the natural functionality of organelles
• Mimicking the natural functionality of organelles using synthetic mechanisms
• Assembly of SOs that equip the host cell with a non-innate functionality

Images of the experimental setup, and the pressure-driven flow controller are shown below:

Key Findings

The droplet-based microfluidic devices were constructed from PDMS, using photo- and soft-lithography methods.^[4] The Elveflow OB1 MK3+ microfluidic flow control system was used to regulate water and oil flow rates precisely.

For the formation of dsGUVs within microfluidic droplets, a final lipid concentration of 1.5 × 10−3 M was employed. The SUV solution was introduced into the aqueous channel of the microfluidic chip for droplet generation. GUVs were produced using 1.25 × 10−3 M PFPE (7000 g mol-1)-PEG(1500 g mol-1)-PFPE(7000 g mol-1) triblock surfactant dissolved in FC-40 oil.

A water-to-oil phase ratio of ≈1:4 was employed for droplet generation. 10 μm diameter droplets were formed at the flow-focusing junction and collected from the outlet of the microfluidic device into a microcentrifuge tube. Video 2 demonstrates the microfluidic setup used for this research. For more details regarding the experimental protocol, please refer to the original article.

Microfluidics based GUV assembly was successfully used to fabricate functional synthetic organelles and exhibit their benefits in different mammalian cells types, including primary cells. Three approaches were investigated and detailed below. Figure 1 shows the schematic representations of all three procedures. Furthermore, Figure 2 shows the principal results obtained through these three synthetic organelle fabrication procedures.

GUV-based Synthetic Organelles are demonstrated to be equipped with diverse operational modules that mimic natural organelle structure and function inside of living cells. Additionally, making use of droplet-based microfluidics for the production of lipid enclosed Synthetic Organelles is a crucial advancement.

Another important result was the foundational approach of a high throughput microfluidic production pipeline, that allowed the researchers to “transfect” millions of cells with Synthetic Organelles in vitro. This will pave the way towards equipment of cell cultures used in industrial production plants with Synthetic Organelles.

These promising results have been achieved with the help of droplet-based microfluidics and the accuracy of a pressure-driven flow controller. For more insight into the research, please refer to the original paper available here.

References