What is Single Cell Injection?
Single cell injection involves delivering substances such as DNA, RNA, proteins, or small molecules directly into individual cells to study their effects on cellular processes. This method is widely used in cell biology for gene function analysis, in genetics for genome editing, in drug development for screening potential therapeutics, and in regenerative medicine for stem cell modification. The ability to manipulate single cells with high precision is critical for understanding complex biological mechanisms and developing new therapies. Over the years, various techniques were developed but efficiency and cell viability issues remained, making innovations like FluidFM essential for advancing single cell research.
Challenges in Single-cell Injection Techniques
From Micro-injection to FluidFM Nano-injection
Micro-injection is part of the current tools available to realize physical delivery into cells alongside with alternative physical transfection methods. Micro-injection showed a certain number of advantages compared to other techniques due to the precision of delivery dosage and timing, the high efficiency of transfection and the low cytotoxicity. Nevertheless, some important drawbacks were found, such as the laborious and slow processes which caused a lower throughput of transfected cells. [1]
Despite those limitations, microinjection has been employed extensively in research to inject various types of biological samples. In the 1980s, J.E. Celis (1984) investigated the process of microinjection into cells with micropipettes and compared it with other transfer techniques. [2] Over the years, several microinjection protocols and setup have been investigated. More than twenty years later, Yan Zhang (2007) proposed a single-cell microinjection technique to both attached and suspended cells, comprising primary cells, cell lines and protozoan. [3] Despite several efforts made to decrease cellular stress and improve the delivery efficiency, the microinjection presented still important drawbacks. But the fast development of fields such as microfluidics, improved the process of microinjection into a single cell.
A. Andamo and K. Jensen (2008) went beyond the limit of traditional methods by reporting a single-cell microinjection in which fluid streams direct into a cell onto a fixed microneedle. This proposal simplified the process and developed the potential of flow through automated microinjection of cells. [4] The precision and control over the injected volumes improved over time. Y. Chow et al., (2016) pushed further the technique by developing the concept of precise microinjection with a quantitatively controlled injection volume based on injection pressure and time. [5] Nowadays, microinjection is still employed to study cell division of mammalian cell culture. C. Day and E. Hinchcliffe (2022) described a microinjection setup to analyze mammalian cells in mitosis with same cell live and fixed imaging. [6]
Moving away from microinjection, the term “nano-injection” was reported in the literature to describe a broad range of methods from a targeted multiphoton optoporation of vital cells [7], a silicon microchip “nano-injector” composed of a microelectromechanical system with an electrically conductive lance [8], also termed Lance Array Nano-injection (LAN) [9], to a hybrid microfluidic chip with a true 3-dimensional nano-injection structure for precise direct delivery of biomolecule into single cells [10]. With the evolution of the instrumentation, the nano-injection method became more and more accurate and controlled, to reach a peak with the development of the FluidFM technology.
Example of direct intra-nuclear nano-injection into mouse primary hepatocyte injected with CRISPR-Cas9 RNP complexes.
FluidFM-Enabled Single Cell Injection
FluidFM (Fluidic Force Microscopy) is an advanced technology that integrates microfluidics with Atomic Force Microscopy (AFM) to enable precise manipulation and injection at the single-cell level. By combining the physical properties of AFM with the versatility of fluidics, FluidFM allows researchers to perform highly controlled single cell nano-injections. The technology involves a hollow cantilever with a nanoscale aperture that can inject or aspirate minute volumes of fluid, ensuring minimal disruption to the cell's natural state. This setup enables direct intra-nuclear or cytoplasmic delivery of reagents without the need for vectors, reducing the risks associated with traditional transfection methods and increasing precision. FluidFM's high accuracy, minimal cell damage, and vector-free delivery make it a superior choice for single cell injection applications.
Applications of FluidFM-enabled Single Cell Injection
Nano-injection into HeLa cells
Here, CRISPR-Cas9 ribonucleoproteic (RNP) complexes were injected in HeLa cells. The cells were monitored over 5 hours, prior to and after the nano-injection of the complex.
Example of nano-injection into adherent cells. HeLa cells, throughput up to 200 cells injected per hour could be achieved.
Examples of nano-injection into adherent cells. CRISPR-Cas9 ribonucleoproteic (RNP) complexes injected in HeLa cells.
Top left:30 min post injection. Top right:1h post injection. Bottom left: 2h post injection. Bottom right: 4h post injection. In the case of HeLa cells, throughput up to 200 cells injected per hours, could be achieved.
Example of nano-injection into adherent cells. Injection of Cas9-GFP protein in Human iPS cell nucleus.
Nano-injection of proteins into cells
Many types of compounds can be injected into cells using the FluidFM technology. Find below an example of a nano-injection of Cas9-GFP protein in Human iPS cell nucleus. A full experimental protocol was prepared, and the experiment performed in-house with the FluidFM OMNIUM. For the full experimental protocol, click here.
Cytoplasmic & nuclear nano-injection into mouse primary hepatocyte
Two types of nano-injections into a mouse primary hepatocyte with CRISPR-Cas9 RNP complexes could be achieved with the FluidFM technology. Both nuclear and cytoplasmic direct nano-injection could be performed.
Example of cytoplasmic nano-injection into Mouse primary hepatocyte injected with CRISPR-Cas9 RNP complexes.
Nano-injection into single fungal cells
Fungal cells represent a challenge for intracellular injection and extraction due to their cell wall. Up to now, the most popular techniques for intracellular delivery into fungi relied on the first breakdown of the cell wall to produce protoplasts, a process that is extremely time-consuming, inefficient, inconsistent, and detrimental to cell survival. [11] Guillaume-Gentil and Orane, et al. (2022) employed the FluidFM technology to inject various solutions into and extract cytoplasmic fluid from individual fungal cells, including unicellular model yeasts and multicellular filamentous fungi. The FluidFM technology offered a strain-free and cargo-independent approach for manipulating and analyzing fungi. [11]
Nano-injection into organelles
Recently, Gäbelein, Christoph G., et al. (2022) proposed a FluidFM-based approach to extract, inject, and transplant organelles from and into living cells with subcellular spatial resolution. Upon the extraction of a set number of mitochondria, a morphological transformation was observed. A pearls-on-a-string phenotype was obtained due to locally applied fluidic forces. mitochondria. With this work, the transplantation of healthy and drug-impaired mitochondria into primary keratinocytes became possible and enabled the monitoring of mitochondrial subpopulation rescue. [12]