Innovative Biotech Solutions

BEGINNER FAQ

Q1. What forms does GelMA come in and what are they for?

Solid GelMA
- Typically a lyophilized (freeze-dried) white foam/powder.
- Long-term storage, shipping, and easy weighing.
Liquid GelMA solution
- GelMA dissolved in aqueous buffer (commonly PBS) at a defined w/v % (e.g., 5–15%).
- Used directly for casting, coating, or as a bioink for bioprinting once photoinitiator is added.

Q2. How do I store GelMA in solid and liquid form?

Solid (lyophilized) GelMA
- Keep dry, protected from light, tightly capped.
- Store at room temperature or 4°C; avoid humidity to prevent clumping and degradation.
Liquid GelMA (no cells)
- Short term (hours–2 days): 4°C (it will gel at low temp; re-melt at 37°C to use).
- Longer term: –20°C or below, aliquoted, then thawed as needed.
Cell-laden GelMA
- Keep in the incubator (37°C, 5% CO₂) after crosslinking and medium exchange.

Q3. How do I turn solid GelMA into a usable liquid solution?

A simple, paper-aligned recipe:

Weigh solid GelMA
- Typical working range: 5–15% (w/v) in PBS or cell culture medium.
- The paper uses 10% (w/v) GelMA extensively as a standard bioink.
Dissolve
- Warm buffer/medium to 37–50°C.
- Add GelMA slowly while stirring until fully clear (no visible particles).
Add photoinitiator
- In the study, LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) at 0.5% (w/v) was used for UV (365 nm) crosslinking.
Filter sterilize if needed
- Pass through 0.22 µm filter while warm (for acellular use).

Now you have a liquid GelMA stock that can be used for casting or loaded into a cartridge for printing.

Q4. What basic temperatures should I think about for GelMA?

37–40°C:
- GelMA is fully liquid, easy to pipette and mix with cells or factors.
~15–25°C (room–cooled):
- GelMA starts to gel physically (coil → triple-helix transitions), increasing viscosity and stabilizing filaments for printing.
<10°C:
- For 10% GelMA, becomes too stiff to extrude (nozzle clogging / non-extrudable).

Q5. How do I make GelMA solid again after it has been melted?

GelMA is thermoresponsive:

If your GelMA solution solidifies in the fridge or on the bench:
- Place the tube/syringe in a 37°C water bath.
- Gently invert or stir until homogeneous.
This is reversible as long as you don’t overheat or over-expose to UV.

Q6. What’s the simplest way to crosslink GelMA?

For standard 3D hydrogels:

Prepare GelMA + LAP at your desired concentration (e.g. 10% + 0.5% LAP).
Pour or print into your mold or onto your substrate.
Expose to UV 365 nm, ~10–18 mW/cm² for ~60 seconds (as in the paper), or adjust for your light source.

That’s your chemically crosslinked GelMA hydrogel.

⚙️ INTERMEDIATE FAQ – Printability, Temperature Windows & Cell Handling

Q7. What is the “two-step” (thermal + photo) crosslinking strategy and why should I care?

The paper uses a two-step crosslinking approach:

Step 1 – Physical gelation (temperature-controlled)
- Cooling the GelMA in the syringe (down to 10–17.5°C) forms triple-helix physical networks, increasing viscosity and filament stability.
Step 2 – Chemical crosslinking (UV + LAP)
- After extrusion, the structure is permanently fixed by UV-induced methacrylate crosslinking.

Why it matters:

Better filament formation and print fidelity before UV.
Tunable mechanical properties, porosity, and degradation via extrusion temperature history.

Q8. What GelMA concentrations and temperatures print well?

From the paper’s systematic printability study (5, 10, 15% GelMA; 5–RT °C; 260 µm nozzle):

<10°C:
- All concentrations (5, 10, 15%) → mostly non-extrudable or severely clogged.
5% GelMA
- 10–12.5°C → irregular, unstable filaments.
- 15°C → can form filaments, but they lose integrity on the collector and bleed at higher temps.
- 17.5°C → too liquid; strong bleeding.
10% GelMA (sweet spot in this system)
- 10°C → thin but less consistent filament.
- 12.5°C → lumpy/unstable.
- 15°C → “printable”: uniform filaments, good structural integrity, moderate width (~500–680 µm from a 260 µm nozzle).
- 17.5°C → more irregular deposition, some bleeding.
- Room temp (~22°C) → bleeding and poor fidelity.
15% GelMA
- Severe clogging at low temperatures; irregular filaments even at higher temps.

Practical takeaway:
For extrusion bioprinting using 10% GelMA in a setup similar to the paper:

Aim for syringe temperature ≈ 15°C to balance:
- Extrudability
- Filament uniformity
- Reasonable fidelity without extreme stiffness

Q9. How does extrusion temperature affect mechanical properties & porosity?

For 10% GelMA extruded at different temperatures then UV-crosslinked:

As extrusion temperature decreases, you get:
- Higher stiffness (E1 from ~21 kPa at RT to ~89 kPa at 10°C).
- Higher viscosity parameter (µ) – more viscoelastic resistance.
- Higher storage modulus (G′) – more elastic behavior.
- Smaller pores & lower porosity
  - Pore size shrinks from ~480–640 µm at RT to ~7–19 µm at 10–12.5°C.
  - Porosity drops from ~50% (bulk) to ~0.2% at 10°C.
- Lower swelling ratios
  - From ~11 (bulk) down to ~2–3 at 10–12.5°C.

So, colder extrusion → denser, stiffer, less swelling, slower-degrading hydrogel.

Q10. How does extrusion temperature affect degradation?

In collagenase II (10 U/mL) over 7 days, 10% GelMA showed:

Bulk / RT-extruded samples → almost fully degraded within ~48 hours.
17.5°C / 15°C extruded → degraded ~93% / 87% by day 7.
12.5°C / 10°C extruded → only ~25% / 13% degraded after 7 days.

Rule of thumb:

Higher extrusion temperature → faster degradation.
Lower extrusion temperature → more stable scaffold (denser network, fewer unreacted C=C bonds, deeper crosslinking).

Q11. What about cell viability in cell-laden GelMA?

Using human dermal fibroblasts in 10% GelMA + 0.5% LAP, printed at different temperatures then UV-crosslinked:

Non-extruded GelMA (just encapsulated + UV)
- ~97% viable at 1 h, ~79–82% at 24 h.
Extruded at room temperature
- ~80% viable at 1 h, ~76% at 24 h.
Extruded at 17.5°C & 15°C
- ~73–77% viable at 24 h (moderate drop vs control).
Extruded at 12.5°C & 10°C
- Severe viability loss: down to ~9–14% viable cells at 24 h.

Interpretation:

Extrusion itself (shear) reduces viability somewhat.
Very low extrusion temperatures (≤12.5°C) → high shear & ultra-dense networks → poor cell survival.

Practical sweet spot for cell-laden extrusion in this system:

10% GelMA with syringe temperature ~15–17.5°C:
- Good printability and acceptable cell viability.

Q12. How should I mix cells into GelMA in practice?

Warm GelMA + LAP to ~37°C so it is fully liquid.
Spin down and resuspend cells to desired density (e.g. 5×10⁵ cells/mL in the study).
Gently mix cells into GelMA using a wide-bore pipette or slow swirling.
Load into a pre-warmed syringe, then cool the syringe to your target extrusion temperature (e.g. 15°C).
Print quickly and crosslink, then add fresh medium.

Keep the time that cells spend in the high-viscosity, cooled state as short as possible to reduce shear damage.

🧪 ADVANCED FAQ – Fine-Tuning Print Fidelity, Temperature, and Structure

Q13. What nozzle and surface conditions improve filament quality?

The paper systematically investigates nozzle hydrophobicity and substrate contact angle with CFD modeling and experiments:

Hydrophobic nozzle (silanized metal, contact angle ≈ 119–160°)
- Reduces adhesion of GelMA to the nozzle.
- Produces thinner, more uniform jets (diameter ~11–12% smaller than hydrophilic nozzle across velocities).
- Increases jet velocity by up to ~20–40% at higher viscosities.
Substrate contact angle ~110° (moderately hydrophobic stage)
- Provides better print fidelity (good symmetry, limited spreading).
- Too hydrophilic (10–60°): excessive spreading and larger printed area.
- Too hydrophobic (160°): unstable lamella, poor support for additional layers.

Takeaway for advanced users:

Use silanized (hydrophobic) nozzles and tune your stage coatings towards ~90–110° contact angle for best filament control.

Q14. How does temperature influence crosslinking chemistry (CD, DSC, FTIR insights)?

Key physicochemical findings:

Circular Dichroism (CD)
- Shows formation of triple helices at lower temperatures for both gelatin and GelMA.
- These helices support reversible physical gelation (thermal step).
Differential Scanning Calorimetry (DSC)
- Gelation transition for GelMA solution around ~15°C (vs ~20°C for gelatin).
- Confirms temperature-dependent coil → helix transition.
FTIR
- Shoulder peak at ~1640 cm⁻¹ (unreacted C=C bonds) progressively disappears at lower extrusion temperatures, and is essentially gone at 10°C.
- Indicates higher crosslinking density (fewer unreacted methacrylate groups) when printing at lower temperatures.

Design implication:

Lower extrusion temperature → more physical pre-ordering → deeper/more complete photopolymerization → stiffer, more stable network.

Q15. How can I deliberately tune stiffness, porosity and degradation using extrusion temperature?

For 10% GelMA + 0.5% LAP (you can generalize the trends to similar systems):

If you want soft, porous, faster-degrading hydrogels:
- Use higher extrusion temperatures / simple casting near room temperature.
- Expect larger pores (~400–600 µm), higher swelling, faster enzymatic breakdown.
If you want stiff, dense, slow-degrading scaffolds:
- Use colder extrusion (e.g., 12.5–15°C).
- Smaller pores, reduced swelling, slower degradation.
- Be careful with cells below ~15°C due to viability drop.
If you want extreme alignment or anisotropic structures:
- At 10–12.5°C, SEM shows elongated, worm-like pore structures and aligned wrinkles, potentially useful for vascular or neural guidance.
- But cell viability is poor at these conditions; better suited for acellular or post-seeded scaffolds.

Q16. How does this translate into concrete “recipes” for different applications?

Example 1 – Cell-laden soft tissue model (e.g., dermal, generic soft tissue)

10% GelMA + 0.5% LAP.
Syringe: 15–17.5°C.
Nozzle: hydrophobic, ~260 µm.
UV: 365 nm, ~1 min at ~10–18 mW/cm².
Expected: good printability, moderate stiffness, cell viability >70% at 24 h.

Example 2 – Long-lasting, stiff scaffold (acellular or post-seeded)

10–15% GelMA + 0.5% LAP.
Syringe: 12.5–15°C (or even 10°C if your system can extrude it).
Same UV conditions.
Expected: dense network, small pores, low swelling, very slow degradation and high modulus; less suitable for direct in-nozzle cell encapsulation.

Example 3 – Fast-degrading, highly porous matrix (e.g., for rapid remodeling)

5–10% GelMA; extrusion at room temperature or gentle casting.
Lower crosslinking density and larger pores; will degrade rapidly in protease-rich environments.

Q17. How does degree of methacrylation (DoM) interact with all this?

The paper used GelMA with ~72–76% methacrylation (measured by ¹H NMR), which gives a fairly high density of photocrosslinkable groups.

Higher DoM → more crosslinking potential → amplifies the effect of low-temperature extrusion (very stiff, dense gels).
Lower DoM → softer, more degradable gels; temperature tuning still works but with a narrower stiffness range.

For an advanced product line, you can combine DoM control + extrusion temperature to create a matrix of formulations covering very soft/fast-degrading to very stiff/slow-degrading, all using the same basic chemistry.

Getting Started with GelMA

Peer-Reviewed Papers

GelMA

Role of temperature on bio-printability of gelatin methacryloyl bioink in two-step cross-linking strategy for tissue engineering applications

M Janmaleki, J Liu, M Kamkar, M Azarmanesh, U Sundararaj, AS Nezhad Biomedical Materials 16 (1), 015021

Covalently cross‐linked hydrogels: Mechanisms of nonlinear viscoelasticity

M Kamkar*, M Janmaleki*, E Erfanian, A Sanati‐Nezhad, U SundararajThe Canadian Journal of Chemical Engineering

Engineering a 3D Human Intracranial Aneurysm Model using Liquid-Assisted Injection Molding and Tuned Hydrogels

KW Yong*, M Janmaleki*, M Pachenari, AP Mitha, A Sanati-Nezhad, ...Acta Biomaterialia

A tuned gelatin methacryloyl (GelMA) hydrogel facilitates myelination of dorsal root ganglia neurons in vitro

S Shahidi*, M Janmaleki*, S Riaz, AS Nezhad, N Syed Materials Science and Engineering: C, 112131

Viscoelastic behavior of covalently crosslinked hydrogels under large shear deformations: An approach to eliminate wall slip

M Kamkar*, M Janmaleki*, E Erfanian, A Sanati-Nezhad, U Sundararaj Physics of Fluids 33 (4), 041702

Microfluidics

Double emulsion formation through hierarchical flow-focusing microchannel

M Azarmanesh, M Farhadi, P Azizian Physics of Fluids 28 (3)

Passive microinjection within high-throughput microfluidics for controlled actuation of droplets and cells

M Azarmanesh, M Dejam, P Azizian, G Yesiloz, AA Mohamad, ...
Scientific reports 9 (1), 6723

Electrohydrodynamic formation of single and double emulsions for low interfacial tension multiphase systems within microfluidics

P Azizian, M Azarmanesh, M Dejam, M Mohammadi, M Shamsi, ...
Chemical Engineering Science 195, 201-207

IVF - High-Throughput Invitro Fertilization