Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Drug Delivery

Drug Delivery - Poster Session D

Poster X11 - A microfluidic device for the cellular loading of small, naïve immune cells

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Adoptive cell transfer (ACT), or the infusion of live cells into a patient for therapeutic treatment, has shown promising results in the clinic in a variety of diseases. However, several challenges preclude ACT from being used widely in the clinic. ACT therapies that rely on ex vivo manipulation of patient cells, such as CAR T therapy, suffer from variable transfection efficiency, which likely contributes to varied response rates. Alternatively, allogeneic ACT, which promises to address sourcing challenges associated with autologous ACT, suffers from high manufacturing costs and limited long-term storage stability.
We have recently developed and published a method of intracellular delivery that relies on veloporation, or the mechanoporation of cell membranes based on viscoelastic stretching. This platform allows for incredibly high-throughput cell loading of human primary T cells, validated with several biomolecules including complex sugars, mRNA, protein, and CRISPR-Cas9 ribonucleoprotein complexes. However, in preliminary studies, the same veloporation device is not as efficacious in smaller immune cells, less than 10 µm, which encompasses many relevant populations including NK cells, neutrophils, naïve T cells, and naïve B cells. These cells have also been historically challenging to manipulate using traditional transfection methods. Here, we aim to adapt the veloporation device to achieve enhanced intracellular delivery in small immune cells.

Materials and

Methods:  A master mold of the CAD-designed devices (Fig. 1A) was made by photolithographic patterning of spin coated SU-8 films on silicon wafers. PDMS replica molds were made from the master mold and cured overnight. The surface of the replica mold was then passivated to allow PDMS to be poured on top to create a secondary mold. Two-part epoxy was mixed and poured into the secondary mold, partially cured for 24 hours, then bonded onto a glass slide. The devices then fully cured for another 24 hours.
The loading media was prepared with 0.5 – 1 mg/ml polyethylene oxide (PEO) and 4kDa FITC dextran in PBS. The cells in this solution were loaded into the devices and pushed through the chip using an air compressor at 6 – 8 bar (Fig. 1B). Cells were recovered and stained with the indicated markers and run on flow cytometry.

Results, Conclusions, and Discussions: Naïve neutrophils were isolated from human donors and prepared in the loading media described above. We tested two device configurations with differing pore sizes, 50µm x 50µm and 30µm x 100µm. We also varied the pressure used to induce fluid flow, from 6 to 8 bar, and the viscosity of the fluid, using 0.5 or 1 mg/ml of PEO. The 50µm x 50µm configuration led to the highest loading efficiency, with about 70% of cells loaded with the FITC dextran (Fig. 1C). Previously, we have seen that increasing the viscosity of the loading media increased the loading efficiency at the cost of cell viability. Here, we indeed experienced significant cell loss using 1 mg/ml PEO. Similarly, increasing the pressure can improve the loading. We saw the most number of loaded cells using 8 bar without significant cell loss. In addition to assessing cargo loading, we analyzed two neutrophil activation markers, CD11b and CD66b. Both markers were expressed at higher levels in loaded cells, indicating that mechanoporation may also lead to neutrophil activation (Fig. 1C).
The technology developed here has wide-spread applications in the realm of immunotherapy and beyond. Many cell types have been incredibly challenging to transfect, which has hindered the field of engineered cell therapy. Even beyond popular reasons for cell transfection, such as CAR engineering, cryopreservation of clinically-relevant immune cells has been equally challenging due to osmotic stress on delicate cell membranes. The technology developed here has the ability to not only improve cell transfection, but also the preservation of cells, bringing ACT closer to an “off-the-shelf” treatment.

Acknowledgements (Optional):