Skip to content
Brilliant Blue Biosciences logoBrilliant BlueBiosciences
Physical methods

Physical methods

Physical methods bypass chemistry entirely and force cargo through the membrane with an electric field, a mechanical impulse or a micro-scale injection. They are the only reliable route into many primary and non-dividing cells, and they are the backbone of therapeutic ex vivo engineering.

5
Candidates profiled with manufacturer protocols
5
Manufacturers whose documentation was used
9
References listed at the end of this page
How it works

From the tube to the target

  1. 1

    Electroporation

    A brief electric pulse of 200–1000 V/cm creates transient hydrophilic pores in the lipid bilayer. Small-molecule probes equilibrate within milliseconds; macromolecules such as DNA and RNP require the pulse plus a stabilising recovery medium.

  2. 2

    Nucleofection

    A vendor-defined combination of buffer, pulse shape and cuvette geometry that drives nucleic acid not just into the cytoplasm but through the nuclear envelope — the reason editing in resting T cells becomes feasible.

  3. 3

    Microinjection

    Direct pressure injection into the nucleus or cytoplasm with a femtotip. Perfect control and 100% certainty of delivery, but serial and low-throughput — used mainly for zygotes and transgenesis.

  4. 4

    Mechanically assisted routes

    Sonoporation uses ultrasound-driven microbubble collapse; magnetofection pulls magnetic nanoparticle–DNA complexes onto cells with a field; hydrodynamic delivery works at organ scale in vivo.

MethodRepresentative platformsTypical use
Electroporation (bulk)Bio-Rad Gene Pulser, BTX, Nepa GeneBacterial and large mammalian batches, DNA
NucleofectionLonza 4D-Nucleofector, SF and SE cell line kitsPrimary T cells, iPSC, NK cells, RNP
High-throughput electroporationNeon, MaxCyte, CellectaScreening in 96- and 384-well
MicroinjectionEppendorf FemtoJet, NarishigeZygotes, transgenesis, single-cell work
MagnetofectionOZ Biosciences Mag4U, ChemicellAdherent cells, low-toxicity local delivery
5 candidates

Reagents and their manufacturer protocols

Each entry below gives the chemistry, the applications the manufacturer validates it for, the dosing basis and the step-by-step protocol. Scale factors follow the manufacturer’s own tables wherever they publish them.

Candidate 01

Lonza Nucleofector (4D / SE cell line kits)

Lonza

V4XC-2032 (4D-Nucleofector X Unit)
Chemistry
Buffered electroporation with optimised pulse programs — combicurve
Best for
Primary human T cells, activated and resting; iPSC and ESC; primary NK cells; CRISPR RNP in immune cells

Dosing basis: Program and buffer are chosen by cell type, not by payload; cargo dose is 20 pmol sgRNA with 10–20 µg Cas9 per 1 × 10⁵–10⁶ cells.

Manufacturer protocol — step by step

  1. 1

    Harvest cells in log phase and count; centrifuge 90 × g for 10 min at room temperature for primary cells.

  2. 2

    Resuspend the pellet in the cell-type-specific Nucleofector solution at the concentration given in the kit protocol (typically 1 × 10⁶ cells per 20 µL).

  3. 3

    Assemble the RNP or DNA at the required dose in the same volume; mix gently and incubate 5–10 min.

  4. 4

    Transfer to the nucleocuvette strip, avoiding bubbles, and select the validated program for the cell type.

  5. 5

    Immediately add 80 µL of pre-warmed, cytokine-containing medium and recover 10 min at 37 °C.

  6. 6

    Transfer to culture at the appropriate density; assess editing at 48–72 h and viability at 24 h.

Protocol notes

  • Pre-warm medium and never leave cells in the Nucleofector solution longer than the protocol allows.
  • For T cells, activation status changes the optimal program — validate for resting versus CD3/CD28-activated cells.
  • RNP delivery outperforms mRNA and plasmid formats for both editing efficiency and viability.

Source: Lonza — 4D-Nucleofector cell-type specific protocols

Candidate 02

Neon Transfection System

Thermo Fisher / Invitrogen

MPK5000
Chemistry
Pipette-tip electroporation with three programmable pulses
Best for
Flexible, small-scale electroporation of difficult lines, primary cells and CRISPR RNP in 10 µL tips

Dosing basis: 1 × 10⁵ cells per 10 µL tip with 0.5–2 µg plasmid DNA or 10–20 pmol RNP.

Manufacturer protocol — step by step

  1. 1

    Wash cells with PBS and resuspend in Buffer R at 1–5 × 10⁷ cells/mL.

  2. 2

    Mix the cell suspension with DNA or RNP in a low-binding tube.

  3. 3

    Draw 10 µL into the Neon tip without bubbles and place in the electroporation chamber filled with Buffer E.

  4. 4

    Apply the validated program for the cell type — typically one to three pulses of 1000–1600 V for 10–30 ms.

  5. 5

    Immediately transfer the tip contents into pre-warmed complete medium and recover overnight before analysis.

Protocol notes

  • Very low volume means low reagent cost per condition — efficient for screening many guides or constructs.
  • Pulse programs must be re-validated for each cell type; published values are starting points only.

Source: Thermo Fisher — Neon transfection system protocols

Candidate 03

MaxCyte flow electroporation

MaxCyte

GT / STx processing assemblies
Chemistry
Continuous-flow electroporation in a closed, single-use processing assembly
Best for
Large-scale ex vivo engineering for therapy — CAR-T manufacturing at 10⁹–10¹⁰ cells

Dosing basis: Scaled to cell number; the platform's purpose is to hold the same electric field dose as the volume increases.

Manufacturer protocol — step by step

  1. 1

    Equilibrate cells in the vendor's electroporation buffer at the process-defined cell density.

  2. 2

    Prepare the payload — mRNA or RNP — at the validated concentration.

  3. 3

    Load the single-use processing assembly and flow cells through the electric field at the set rate.

  4. 4

    Collect the electroporated product directly into pre-warmed recovery medium without an open transfer step.

  5. 5

    Incubate for the validated recovery period before downstream processing.

Protocol notes

  • The closed, single-use assembly is the design feature that makes it compatible with clinical manufacturing.
  • Electrical dose must be defined per cell type and payload; the engineering holds it constant as you scale.

Source: MaxCyte — ExPERT flow electroporation platform

Candidate 04

Microinjection

Eppendorf / Narishige / Sutter

FemtoJet 4i
Chemistry
None — direct mechanical delivery
Best for
Zygote and embryo transgenesis, CRISPR knock-in in single cells, and quantitative delivery where every cell must receive an exact dose

Dosing basis: Femto­litre to picolitre injection volumes, calibrated per needle and per cell type.

Manufacturer protocol — step by step

  1. 1

    Pull and bevel the injection needle; back-fill with the cargo solution and mount in the micromanipulator.

  2. 2

    Place the specimen (zygote, embryo, adherent cell) in a drop of medium under mineral oil on an inverted microscope.

  3. 3

    Hold the cell with the holding pipette and pierce the membrane with the injection needle.

  4. 4

    Inject the calibrated volume into the cytoplasm or pronucleus depending on the goal.

  5. 5

    Withdraw and culture; survival rates above 80% indicate the needle and pressure are tuned correctly.

Protocol notes

  • The only method that guarantees delivery to every treated cell, which is why it is standard for transgenesis.
  • Throughput is limited and the technique requires dedicated micromanipulation training.

Source: Eppendorf — Microinjection application notes

Candidate 05

Magnetofection

OZ Biosciences (Mag4U), Chemicell

OZ Biosciences Mag4U
Chemistry
Magnetic nanoparticle–DNA complexes pulled into cells by a magnetic field
Best for
Adherent cells and localised in vivo delivery where low toxicity and short exposure matter

Dosing basis: Associates DNA with magnetic nanoparticles, then applies the field; dose scales with cell number and field strength.

Manufacturer protocol — step by step

  1. 1

    Associate the nucleic acid with the magnetic nanoparticle reagent according to the vendor ratio.

  2. 2

    Add the complex to cells and place the magnetic plate beneath the culture vessel.

  3. 3

    Incubate 15–30 min with the field applied — uptake is field-driven and much faster than passive endocytosis.

  4. 4

    Remove the magnet, wash free complex away and replace with complete medium.

  5. 5

    Assay at 24–48 h.

Protocol notes

  • Short exposure with a field keeps free reagent contact minimal, which lowers apparent toxicity.
  • Useful when a strong lipid reagent is cytotoxic in the target culture.

Source: OZ Biosciences — Magnetofection technology

Cross-cutting rules

What applies to every candidate on this page

  • Programs and buffers are cell-type specific; a published pulse value is a starting point, not a validated condition.
  • Recovery medium composition and temperature have more effect on viability than the pulse itself in primary cells.
  • Electroporation scales by cell number and constant electric dose, not by volume of reagent.

Let's engineer the next delivery breakthrough together

We co-develop nanocarrier and biosensing programmes with pharma, biotech and academic groups — from target selection through GMP supply.

ResearchersRequest a technical consultationTalk formulation, characterisation or assay design with one of our scientists.Industry partnersExplore co-developmentFeasibility studies, co-development programmes, licensing and GMP supply.InvestorsDownload our capabilities deckPlatform, pipeline milestones, manufacturing and partnership models in one PDF.